2&#39; and 3&#39;-nucleoside prodrugs for treating Flaviviridae infections

ABSTRACT

2′ and 3′-Prodrugs of 1′, 2′, 3′ or 4′-branched β-D or β-L nucleosides, or their pharmaceutically acceptable salts and derivatives are described, which are useful in the prevention and treatment of Flaviviridae infections and other related conditions. These modified nucleosides provide superior results against flaviviruses and pestiviruses, including hepatitis C virus and viruses generally that replicate through an RNA dependent RNA reverse transcriptase. Compounds, compositions, methods and uses are provided for the treatment of Flaviviridae infection, including HCV infection, that include the administration of an effective amount of the prodrugs of the present invention, or their pharmaceutically acceptable salts or derivatives. These drugs may optionally be administered in combination or alteration with further anti-viral agents to prevent or treat Flaviviridae infections and other related conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/609,298, filed Jun. 27, 2003, which claims the benefit of priority toU.S. Provisional application No. 60/392,351, filed Jun. 28, 2002; U.S.Provisional Application No. 60/466,194, filed Apr. 28, 2003; and U.S.Provisional application 60/470,949, filed May 14, 2003, the disclosuresof each of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention is in the area of pharmaceutical chemistry, and is inparticular, a 2′ and/or 3′ prodrug of a 1′, 2′, 3′ or 4′-branchednucleosides for the treatment of a Flaviviridae infection, such as ahepatitis C virus infection.

BACKGROUND OF THE INVENTION

Flaviviridae Viruses

The Flaviviridae family of viruses comprises at least three distinctgenera: pestiviruses, which cause disease in cattle and pigs;flaviviruses, which are the primary cause of diseases such as denguefever and yellow fever; and hepaciviruses, whose sole member is HCV. Theflavivirus genus includes more than 68 members separated into groups onthe basis of serological relatedness (Calisher et al., J. Gen. Virol,1993, 70, 37-43). Clinical symptoms vary and include fever, encephalitisand hemorrhagic fever (Fields Virology, Editors: Fields, B. N., Knipe,D. M., and Howley, P. M., Lippincott-Raven Publishers, Philadelphia,Pa., 1996, Chapter 31, 931-959). Flaviviruses of global concern that areassociated with human disease include the dengue hemorrhagic feverviruses (DHF), yellow fever virus, shock syndrome and Japaneseencephalitis virus (Halstead, S. B., Rev. Infect. Dis., 1984, 6,251-264; Halstead, S. B., Science, 239:476-481, 1988; Monath, T. P., NewEng. J. Med., 1988, 319, 641-643).

The pestivirus genus includes bovine viral diarrhea virus (BVDV),classical swine fever virus (CSFV, also called hog cholera virus) andborder disease virus (BDV) of sheep (Moennig, V. et al. Adv. Vir. Res.1992, 41, 53-98). Pestivirus infections of domesticated livestock(cattle, pigs and sheep) cause significant economic losses worldwide.BVDV causes mucosal disease in cattle and is of significant economicimportance to the livestock industry (Meyers, G. and Thiel, H.-J.,Advances in Virus Research, 1996, 47, 53-118; Moennig V., et al, Adv.Vir. Res. 1992, 41, 53-98). Human pestiviruses have not been asextensively characterized as the animal pestiviruses. However,serological surveys indicate considerable pestivirus exposure in humans.

Pestiviruses and hepaciviruses are closely related virus groups withinthe Flaviviridae family. Other closely related viruses in this familyinclude the GB virus A, GB virus A-like agents, GB virus-B and GBvirus-C (also called hepatitis G virus, HGV). The hepacivirus group(hepatitis C virus; HCV) consists of a number of closely related butgenotypically distinguishable viruses that infect humans. There areapproximately 6 HCV genotypes and more than 50 subtypes. Due to thesimilarities between pestiviruses and hepaciviruses, combined with thepoor ability of hepaciviruses to grow efficiently in cell culture,bovine viral diarrhea virus (BVDV) is often used as a surrogate to studythe HCV virus.

The genetic organization of pestiviruses and hepaciviruses is verysimilar. These positive stranded RNA viruses possess a single large openreading frame (ORF) encoding all the viral proteins necessary for virusreplication. These proteins are expressed as a polyprotein that is co-and post-translationally processed by both cellular and virus-encodedproteinases to yield the mature viral proteins. The viral proteinsresponsible for the replication of the viral genome RNA are locatedwithin approximately the carboxy-terminal. Two-thirds of the ORF aretermed nonstructural (NS) proteins. The genetic organization andpolyprotein processing of the nonstructural protein portion of the ORFfor pestiviruses and hepaciviruses is very similar. For both thepestiviruses and hepaciviruses, the mature nonstructural (NS) proteins,in sequential order from the amino-terminus of the nonstructural proteincoding region to the carboxy-terminus of the ORF, consist of p7, NS2,NS3, NS4A, NS4B, NS5A, and NS5B.

The NS proteins of pestiviruses and hepaciviruses share sequence domainsthat are characteristic of specific protein functions. For example, theNS3 proteins of viruses in both groups possess amino acid sequencemotifs characteristic of serine proteinases and of helicases (Gorbalenyaet al. (1988) Nature 333:22; Bazan and Fletterick (1989) Virology171:637-639; Gorbalenya et al. (1989) Nucleic Acid Res. 17.3889-3897).Similarly, the NS5B proteins of pestiviruses and hepaciviruses have themotifs characteristic of RNA-directed RNA polymerases (Koonin, E. V. andDolja, V. V. (1993) Crit. Rev. Biochem. Molec. Biol. 28:375-430).

The actual roles and functions of the NS proteins of pestiviruses andhepaciviruses in the lifecycle of the viruses are directly analogous. Inboth cases, the NS3 serine proteinase is responsible for all proteolyticprocessing of polyprotein precursors downstream of its position in theORF (Wiskerchen and Collett (1991) Virology 184:341-350; Bartenschlageret al. (1993) J. Virol. 67:3835-3844; Eckart et al. (1993) Biochem.Biophys. Res. Comm. 192:399-406; Grakoui et al. (1993) J. Virol.67:2832-2843; Grakoui et al. (1993) Proc. Natl. Acad. Sci. USA90:10583-10587; Hijikata et al. (1993) J. Virol. 67:4665-4675; Tome etal. (1993) J. Virol. 67:4017-4026). The NS4A protein, in both cases,acts as a cofactor with the NS3 serine protease (Bartenschlager et al.(1994) J. Virol. 68:5045-5055; Failla et al. (1994) J. Virol. 68:3753-3760; Lin et al. (1994) 68:8147-8157; Xu et al. (1997) J. Virol.71:5312-5322). The NS3 protein of both viruses also functions as ahelicase (Kim et al. (1995) Biochem. Biophys. Res. Comm. 215: 160-166;Jin and Peterson (1995) Arch. Biochem. Biophys., 323:47-53; Warrener andCollett (1995) J. Virol. 69:1720-1726). Finally, the NS5B proteins ofpestiviruses and hepaciviruses have the predicted RNA-directed RNApolymerases activity (Behrens et al. (1996) EMBO J. 15:12-22; Lchmann etal. (1997) J. Virol. 71:8416-8428; Yuan et al. (1997) Biochem. Biophys.Res. Comm. 232:231-235; Hagedorn, PCT WO 97/12033; Zhong et al. (1998)J. Virol. 72.9365-9369).

Hepatitis C Virus

The hepatitis C virus (HCV) is the leading cause of chronic liverdisease worldwide. (Boyer, N. et al. J. Hepatol. 32:98-112, 2000). HCVcauses a slow growing viral infection and is the major cause ofcirrhosis and hepatocellular carcinoma (Di Besceglie, A. M. and Bacon,B. R., Scientific American, October: 80-85, (1999); Boyer, N. et al. J.Hepatol. 32:98-112, 2000). An estimated 170 million persons are infectedwith HCV worldwide. (Boyer, N. et al. J. Hepatol. 32:98-112, 2000).Cirrhosis caused by chronic hepatitis C infection accounts for8,000-12,000 deaths per year in the United States, and HCV infection isthe leading indication for liver transplantation.

HCV is known to cause at least 80% of posttransfusion hepatitis and asubstantial proportion of sporadic acute hepatitis. Preliminary evidencealso implicates HCV in many cases of “idiopathic” chronic hepatitis,“cryptogenic” cirrhosis, and probably hepatocellular carcinoma unrelatedto other hepatitis viruses, such as Hepatitis B Virus (HBV). A smallproportion of healthy persons appear to be chronic HCV carriers, varyingwith geography and other epidemiological factors. The numbers maysubstantially exceed those for HBV, though information is stillpreliminary; how many of these persons have subclinical chronic liverdisease is unclear. (The Merck Manual, ch. 69, p. 901, 16th ed.,(1992)).

HCV is an enveloped virus containing a positive-sense single-strandedRNA genome of approximately 9.4 kb. The viral genome consists of a 5′untranslated region (UTR), a long open reading frame encoding apolyprotein precursor of approximately 3011 amino acids, and a short 3′UTR. The 5′ UTR is the most highly conserved part of the HCV genome andis important for the initiation and control of polyprotein translation.Translation of the HCV genome is initiated by a cap-independentmechanism known as internal ribosome entry. This mechanism involves thebinding of ribosomes to an RNA sequence known as the internal ribosomeentry site (IRES). An RNA pseudoknot structure has recently beendetermined to be an essential structural element of the HCV IRES. Viralstructural proteins include a nucleocapsid core protein (C) and twoenvelope glycoproteins, E1 and E2. HCV also encodes two proteinases, azinc-dependent metalloproteinase encoded by the NS2-NS3 region and aserine proteinase encoded in the NS3 region. These proteinases arerequired for cleavage of specific regions of the precursor polyproteininto mature peptides. The carboxyl half of nonstructural protein 5,NS5B, contains the RNA-dependent RNA polymerase. The function of theremaining nonstructural proteins, NS4A and NS4B, and that of NS5A (theamino-terminal half of nonstructural protein 5) remain unknown.

A significant focus of current antiviral research is directed to thedevelopment of improved methods of treatment of chronic HCV infectionsin humans (Di Besceglie, A. M. and Bacon, B. R., Scientific American,October: 80-85, (1999)).

Treatment of HCV Infection with Interferon

Interferons (IFNs) have been commercially available for the treatment ofchronic hepatitis for nearly a decade. IFNs are glycoproteins producedby immune cells in response to viral infection. IFNs inhibit replicationof a number of viruses, including HCV, and when used as the soletreatment for hepatitis C infection, IFN can in certain cases suppressserum HCV-RNA to undetectable levels. Additionally, IFN can normalizeserum amino transferase levels. Unfortunately, the effect of IFN istemporary and a sustained response occurs in only 8%-9% of patientschronically infected with HCV (Gary L. Davis. Gastroenterology118:S104-S114, 2000). Most patients, however, have difficulty toleratinginterferon treatment, which causes severe flu-like symptoms, weightloss, and lack of energy and stamina.

A number of patents disclose Flaviviridae, including HCV, treatments,using interferon-based therapies. For example, U.S. Pat. No. 5,980,884to Blatt et al. discloses methods for retreatment of patients afflictedwith HCV using consensus interferon. U.S. Pat. No. 5,942,223 to Bazer etal. discloses an anti-HCV therapy using ovine or bovine interferon-tau.U.S. Pat. No. 5,928,636 to Alber et al. discloses the combinationtherapy of interleukin-12 and interferon alpha for the treatment ofinfectious diseases including HCV. U.S. Pat. No. 5,849,696 to Chretienet al. discloses the use of thymosins, alone or in combination withinterferon, for treating HCV. U.S. Pat. No. 5,830,455 to Valtuena et al.discloses a combination HCV therapy employing interferon and a freeradical scavenger. U.S. Pat. No. 5,738,845 to Imakawa discloses the useof human interferon tau proteins for treating HCV. Otherinterferon-based treatments for HCV are disclosed in U.S. Pat. No.5,676,942 to Testa et al., U.S. Pat. No. 5,372,808 to Blatt et al., andU.S. Pat. No. 5,849,696. A number of patents also disclose pegylatedforms of interferon, such as U.S. Pat. Nos. 5,747,646, 5,792,834 and5,834,594 to Hoffmann-La Roche Inc; PCT Publication No. WO 99/32139 andWO 99/32140 to Enzon; WO 95/13090 and U.S. Pat. Nos. 5,738,846 and5,711,944 to Schering; and U.S. Pat. No. 5,908,621 to Glue et al.

Interferon alpha-2a and interferon alpha-2b are currently approved asmonotherapy for the treatment of HCV. ROFERON®-A (Roche) is therecombinant form of interferon alpha-2a. PEGASYS® (Roche) is thepegylated (i.e. polyethylene glycol modified) form of interferonalpha-2a. INTRON®A (Schering Corporation) is the recombinant form ofInterferon alpha-2b, and PEG-INTRON® (Schering Corporation) is thepegylated form of interferon alpha-2b.

Other forms of interferon alpha, as well as interferon beta, gamma, tauand omega are currently in clinical development for the treatment ofHCV. For example, INFERGEN (interferon alphacon-1) by InterMune,OMNIFERON (natural interferon) by Viragen, ALBUFERON by Human GenomeSciences, REBIF (interferon beta-1a) by Ares-Serono, Omega Interferon byBioMedicine, Oral Interferon Alpha by Amarillo Biosciences, andinterferon gamma, interferon tau, and interferon gamma-1b by InterMuneare in development.

Ribivarin

Ribavirin (1-β-D-ribofuranosyl-1-1,2,4-triazole-3-carboxamide) is asynthetic, non-interferon-inducing, broad spectrum antiviral nucleosideanalog sold under the trade name, Virazole (The Merck Index, 11thedition, Editor: Budavari, S., Merck & Co., Inc., Rahway, N.J., p 1304,1989). U.S. Pat. No. 3,798,209 and RE29,835 disclose and claimribavirin. Ribavirin is structurally similar to guanosine, and has invitro activity against several DNA and RNA viruses includingFlaviviridae (Gary L. Davis. Gastroenterology 118:S104-S114, 2000).

Ribavirin reduces serum amino transferase levels to normal in 40% ofpatients, but it does not lower serum levels of HCV-RNA (Gary L. Davis.Gastroenterology 118:S104-S 14, 2000). Thus, ribavirin alone is noteffective in reducing viral RNA levels. Additionally, ribavirin hassignificant toxicity and is known to induce anemia.

Ribavirin is not approved for monotherapy against HCV. It has beenapproved in combination with interferon alpha-2a or interferon alpha-2bfor the treatment of HCV.

Combination of Interferon and Ribavirin

The current standard of care for chronic hepatitis C is combinationtherapy with an alpha interferon and ribavirin. The combination ofinterferon and ribavirin for the treatment of HCV infection has beenreported to be effective in the treatment of interferon naïve patients(Battaglia, A. M. et al., Ann. Pharmacother. 34:487-494, 2000), as wellas for treatment of patients when histological disease is present(Berenguer, M. et al. Antivir. Ther. 3(Suppl. 3):125-136, 1998). Studieshave show that more patients with hepatitis C respond to pegylatedinterferon-alpha/ribavirin combination therapy than to combinationtherapy with unpegylated interferon alpha. However, as with monotherapy,significant side effects develop during combination therapy, includinghemolysis, flu-like symptoms, anemia, and fatigue. (Gary L. Davis.Gastroenterology 118:S104-S114, 2000).

Combination therapy with PEG-INTRON® (peginterferon alpha-2b) andREBETOL® (Ribavirin, USP) Capsules is available from ScheringCorporation. REBETOL® (Schering Corporation) has also been approved incombination with INTRON® A (Interferon alpha-2b, recombinant, ScheringCorporation). Roche's PEGASYS® (pegylated interferon alpha-2a) andCOPEGUS® (ribavirin) are also approved for the treatment of HCV.

PCT Publication Nos. WO 99/59621, WO 00/37110, WO 01/81359, WO 02/32414and WO 03/024461 by Schering Corporation disclose the use of pegylatedinterferon alpha and ribavirin combination therapy for the treatment ofHCV. PCT Publication Nos. WO 99/15194, WO 99/64016, and WO 00/24355 byHoffmann-La Roche Inc also disclose the use of pegylated interferonalpha and ribavirin combination therapy for the treatment of HCV.

Additional Methods to Treat Flaviviridae Infections

The development of new antiviral agents for flaviviridae infections,especially hepatitis C, is currently underway. Specific inhibitors ofHCV-derived enzymes such as protease, helicase, and polymeraseinhibitors are being developed. Drugs that inhibit other steps in HCVreplication are also in development, for example, drugs that blockproduction of HCV antigens from the RNA (IRES inhibitors), drugs thatprevent the normal processing of HCV proteins (inhibitors ofglycosylation), drugs that block entry of HCV into cells (by blockingits receptor) and nonspecific cytoprotective agents that block cellinjury caused by the virus infection. Further, molecular approaches arealso being developed to treat hepatitis C, for example, ribozymes, whichare enzymes that break down specific viral RNA molecules, and antisenseoligonucleotides, which are small complementary segments of DNA thatbind to viral RNA and inhibit viral replication, are underinvestigation. A number of HCV treatments are reviewed by Bymock et al.in Antiviral Chemistry & Chemotherapy, 11:2; 79-95 (2000) and DeFrancesco et al. in Antiviral Research, 58: 1-16 (2003).

Examples of classes of drugs that are being developed to treatFlaviviridae infections include:

(1) Protease Inhibitors

Substrate-based NS3 protease inhibitors (Attwood et al., Antiviralpeptide derivatives, PCT WO 98/22496, 1998; Attwood et al., AntiviralChemistry and Chemotherapy 1999, 10, 259-273; Attwood et al.,Preparation and use of amino acid derivatives as anti-viral agents,German Patent Pub. DE 19914474; Tung et al. Inhibitors of serineproteases, particularly hepatitis C virus NS3 protease, PCT WO98/17679), including alphaketoamides and hydrazinoureas, and inhibitorsthat terminate in an electrophile such as a boronic acid or phosphonate(Llinas-Brunet et al, Hepatitis C inhibitor peptide analogues, PCT WO99/07734) are being investigated.

Non-substrate-based NS3 protease inhibitors such as2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al.,Biochemical and Biophysical Research Communications, 1997, 238, 643-647;Sudo K. et al. Antiviral Chemistry and Chemotherapy, 1998, 9, 186),including RD3-4082 and RD3-4078, the former substituted on the amidewith a 14 carbon chain and the latter processing a para-phenoxyphenylgroup are also being investigated.

Sch 68631, a phenanthrenequinone, is an HCV protease inhibitor (Chu M.et al., Tetrahedron Letters 37:7229-7232, 1996). In another example bythe same authors, Sch 351633, isolated from the fungus Penicilliumgriseofulvum, was identified as a protease inhibitor (Chu M. et al.,Bioorganic and Medicinal Chemistry Letters 9:1949-1952). Nanomolarpotency against the HCV NS3 protease enzyme has been achieved by thedesign of selective inhibitors based on the macromolecule eglin c. Eglinc, isolated from leech, is a potent inhibitor of several serineproteases such as S. griseus proteases A and B, α-chymotrypsin, chymaseand subtilisin. Qasim M. A. et al., Biochemistry 36:1598-1607, 1997.

Several U.S. patents disclose protease inhibitors for the treatment ofHCV. For example, U.S. Pat. No. 6,004,933 to Spruce et al. discloses aclass of cysteine protease inhibitors for inhibiting HCV endopeptidase2. U.S. Pat. No. 5,990,276 to Zhang et al. discloses syntheticinhibitors of hepatitis C virus NS3 protease. The inhibitor is asubsequence of a substrate of the NS3 protease or a substrate of theNS4A cofactor. The use of restriction enzymes to treat HCV is disclosedin U.S. Pat. No. 5,538,865 to Reyes et al. Peptides as NS3 serineprotease inhibitors of HCV are disclosed in WO 02/008251 to CorvasInternational, Inc, and WO 02/08187 and WO 02/008256 to ScheringCorporation. HCV inhibitor tripeptides are disclosed in U.S. Pat. Nos.6,534,523, 6,410,531, and 6,420,380 to Boehringer Ingelheim and WO02/060926 to Bristol Myers Squibb. Diaryl peptides as NS3 serineprotease inhibitors of HCV are disclosed in WO 02/48172 to ScheringCorporation. Imidazoleidinones as NS3 serine protease inhibitors of HCVare disclosed in WO 02/08198 to Schering Corporation and WO 02/48157 toBristol Myers Squibb. WO 98/17679 to Vertex Pharmaceuticals and WO02/48116 to Bristol Myers Squibb also disclose HCV protease inhibitors.

-   -   (2) Thiazolidine derivatives which show relevant inhibition in a        reverse-phase HPLC assay with an NS3/4A fusion protein and        NS5A/5B substrate (Sudo K. et al., Antiviral Research, 1996, 32,        9-18), especially compound RD-1-6250, possessing a fused        cinnamoyl moiety substituted with a long alkyl chain, RD4 6205        and RD4 6193;    -   (3) Thiazolidines and benzanilides identified in Kakiuchi N. et        al. J. EBS Letters 421, 217-220; Takeshita N. et al. Analytical        Biochemistry, 1997, 247, 242-246;    -   (4) A phenan-threnequinone possessing activity against protease        in a SDS-PAGE and autoradiography assay isolated from the        fermentation culture broth of Streptomyces sp., Sch 68631        (Chu M. et al., Tetrahedron Letters, 1996, 37, 7229-7232), and        Sch 351633, isolated from the fungus Penicillium griseofulvum,        which demonstrates activity in a scintillation proximity assay        (Chu M. et al., Bioorganic and Medicinal Chemistry Letters 9,        1949-1952);    -   (5) Helicase inhibitors (Diana G. D. et al., Compounds,        compositions and methods for treatment of hepatitis C, U.S. Pat.        No. 5,633,358; Diana G. D. et al., Piperidine derivatives,        pharmaceutical compositions thereof and their use in the        treatment of hepatitis C, PCT WO 97/36554);    -   (6) Nucleotide polymerase inhibitors and gliotoxin (Ferrari R.        et al. Journal of Virology, 1999, 73, 1649-1654), and the        natural product cerulenin (Lohmann V. et al., Virology, 1998,        249, 108-118);    -   (7) Antisense phosphorothioate oligodeoxynucleotides (S-ODN)        complementary to sequence stretches in the 5′ non-coding region        (NCR) of the virus (Alt M. et al., Hepatology, 1995, 22,        707-717), or nucleotides 326-348 comprising the 3′ end of the        NCR and nucleotides 371-388 located in the core coding region of        the HCV RNA (Alt M. et al., Archives of Virology, 1997, 142,        589-599; Galderisi U. et al., Journal of Cellular Physiology,        1999, 181, 251-257);    -   (8) Inhibitors of IRES-dependent translation (Ikeda N et al.,        Agent for the prevention and treatment of hepatitis C, Japanese        Patent Pub. JP-08268890; Kai Y. et al. Prevention and treatment        of viral diseases, Japanese Patent Pub. JP-10101591);    -   (9) Ribozymes, such as nuclease-resistant ribozymes        (Maccjak, D. J. et al., Hepatology 1999, 30, abstract 995) and        those disclosed in U.S. Pat. No. 6,043,077 to Barber et al., and        U.S. Pat. Nos. 5,869,253 and 5,610,054 to Draper et al.; and    -   (10) Nucleoside analogs have also been developed for the        treatment of Flaviviridae infections.

Idenix Pharmaceuticals the use of branched in the treatment offlaviviruses (including HCV) and pestiviruses in InternationalPublication Nos. WO 01/90121 and WO 01/92282. Specifically, a method forthe treatment of hepatitis C infection (and flaviviruses andpestiviruses) in humans and other host animals is disclosed in theIdenix publications that includes administering an effective amount of abiologically active 1′, 2′, 3′ or 4′-branched β-D or β-L nucleosides ora pharmaceutically acceptable salt or derivative thereof, administeredeither alone or in combination with another antiviral agent, optionallyin a pharmaceutically acceptable carrier.

Other patent applications disclosing the use of certain nucleosideanalogs to treat hepatitis C virus include: PCT/CA00/01316 (WO 01/32153;filed Nov. 3, 2000) and PCT/CA01/00197 (WO 01/60315; filed Feb. 19,2001) filed by BioChem Pharma, Inc. (now Shire Biochem, Inc.);PCT/US02/01531 (WO 02/057425; filed Jan. 18, 2002) and PCT/US02/03086(WO 02/057287; filed Jan. 18, 2002) filed by Merck & Co., Inc.,PCT/EP01/09633 (WO 02/18404; published Aug. 21, 2001) filed by Roche,and PCT Publication Nos. WO 01/79246 (filed Apr. 13, 2001), WO 02/32920(filed Oct. 18, 2001) and WO 02/48165 by Pharmasset, Ltd.

PCT Publication No. WO 99/43691 to Emory University, entitled“2′-Fluoronucleosides” discloses the use of certain 2′-fluoronucleosidesto treat HCV.

Eldrup et al. (Oral Session V, Hepatitis C Virus, Flaviviridae; 16^(th)International Conference on Antiviral Research (Apr. 27, 2003, Savannah,Ga.)) described the structure activity relationship of 2′-modifiednucleosides for inhibition of HCV.

Bhat et al. (Oral Session V, Hepatitis C Virus, Flaviviridae, 2003 (OralSession V, Hepatitis C Virus, Flaviviridae; 16^(th) InternationalConference on Antiviral Research (Apr. 27, 2003, Savannah, Ga.); p A75)describe the synthesis and pharmacokinetic properties of nucleosideanalogues as possible inhibitors of HCV RNA replication. The authorsreport that 2′-modified nucleosides demonstrate potent inhibitoryactivity in cell-based replicon assays.

Olsen et al. (Oral Session V, Hepatitis C Virus, Flaviviridae; 16^(th)International Conference on Antiviral Research (Apr. 27, 2003, Savannah,Ga.) p A76) also described the effects of the 2′-modified nucleosides onHCV RNA replication.

(11) Other miscellaneous compounds including 1-amino-alkylcyclohexanes(U.S. Pat. No. 6,034,134 to Gold et al.), alkyl lipids (U.S. Pat. No.5,922,757 to Chojkier et al.), vitamin E and other antioxidants (U.S.Pat. No. 5,922,757 to Chojkier et al.), squalene, amantadine, bile acids(U.S. Pat. No. 5,846,964 to Ozeki et al.),N-(phosphonoacetyl)-L-aspartic acid, (U.S. Pat. No. 5,830,905 to Dianaet al.), benzenedicarboxamides (U.S. Pat. No. 5,633,388 to Diana etal.), polyadenylic acid derivatives (U.S. Pat. No. 5,496,546 to Wang etal.), 2′,3′-dideoxyinosine (U.S. Pat. No. 5,026,687 to Yarchoan et al.),benzimidazoles (U.S. Pat. No. 5,891,874 to Colacino et al.), plantextracts (U.S. Pat. No. 5,837,257 to Tsai et al., U.S. Pat. No.5,725,859 to Omer et al., and U.S. Pat. No. 6,056,961), and piperidenes(U.S. Pat. No. 5,830,905 to Diana et al.).

(12) Other compounds currently in preclinical or clinical developmentfor treatment of hepatitis C virus include: Interleukin-10 bySchering-Plough, IP-501 by Interneuron, Merimebodib (VX-497) by Vertex,AMANTADINE® (Symmetrel) by Endo Labs Solvay, HEPTAZYME® by RPI, IDN-6556by Idun Pharma., XTL-002 by XTL., HCV/MF59 by Chiron, CIVACIR®(Hepatitis C Immune Globulin) by NABI, LEVOVIRIN® by ICN/Ribapharm,VIRAMIDINE® by ICN/Ribapharm, ZADAXIN® (thymosin alpha-1) by Sci Clone,thymosin plus pegylated interferon by Sci Clone, CEPLENE® (histaminedihydrochloride) by Maxim, VX 950/LY 570310 by Vertex/Eli Lilly, ISIS14803 by Isis Pharmaceutical/Elan, IDN-6556 by Idun Pharmaceuticals,Inc., JTK 003 by AKROS Pharma, BILN-2061 by Boehringer Ingelheim,CellCept (mycophenolate mofetil) by Roche, T67, a β-tubulin inhibitor,by Tularik, a therapeutic vaccine directed to E2 by Innogenetics, FK788by Fujisawa Healthcare, Inc., 1 dB 1016 (Siliphos, oralsilybin-phosphatdylcholine phytosome), RNA replication inhibitors(VP50406) by ViroPharma/Wyeth, therapeutic vaccine by Intercell,therapeutic vaccine by Epimmune/Genencor, IRES inhibitor by Anadys, ANA245 and ANA 246 by Anadys, immunotherapy (Therapore) by Avant, proteaseinhibitor by Corvas/SChering, helicase inhibitor by Vertex, fusioninhibitor by Trimeris, T cell therapy by CellExSys, polymerase inhibitorby Biocryst, targeted RNA chemistry by PTC Therapeutics, Dication byImmtech, Int., protease inhibitor by Agouron, protease inhibitor byChiron/Medivir, antisense therapy by AVI BioPharma, antisense therapy byHybridon, hemopurifier by Aethlon Medical, therapeutic vaccine by Merix,protease inhibitor by Bristol-Myers Squibb/Axys, Chron-VacC, atherapeutic vaccine, by Tripep, UT 231B by United Therapeutics,protease, helicase and polymerase inhibitors by Genelabs Technologies,IRES inhibitors by Immusol, R803 by Rigel Pharmaceuticals, INFERGEN®(interferon alphacon-1) by InterMune, OMNIFERON® (natural interferon) byViragen, ALBUFERON® by Human Genome Sciences, REBIF® (interferonbeta-1a) by Ares-Serono, Omega Interferon by BioMedicine, OralInterferon Alpha by Amarillo Biosciences, interferon gamma, interferontau, and Interferon gamma-1b by InterMune.

Nucleoside prodrugs have been previously described for the treatment ofother forms of hepatitis. WO 00/09531 (filed Aug. 10, 1999) and WO01/96353 (filed Jun. 15, 2001) to Idenix Pharmaceuticals, discloses2′-deoxy-β-L-nucleosides and their 3′-prodrugs for the treatment of HBV.U.S. Pat. No. 4,957,924 to Beauchamp discloses various therapeuticesters of acyclovir.

In light of the fact that HCV infection has reached epidemic levelsworldwide, and has tragic effects on the infected patient, there remainsa strong need to provide new effective pharmaceutical agents to treathepatitis C that have low toxicity to the host.

Further, given the rising threat of other flaviviridae infections, thereremains a strong need to provide new effective pharmaceutical agentsthat have low toxicity to the host.

Therefore, it is an object of the present invention to provide acompound, method and composition for the treatment of a host infectedwith flaviviridae, including hepatitis C virus.

It is another object of the present invention to provide a compound,method and composition generally for the treatment of patients infectedwith pestiviruses, flaviviruses, or hepaciviruses.

SUMMARY OF THE INVENTION

2′- and 3′-prodrugs of 1′, 2′, 3′ or 4′-branched β-D or β-L nucleosides,or their pharmaceutically acceptable salts, or pharmaceuticallyacceptable formulations containing these compounds are useful in theprevention and treatment of Flaviviridae infections and other relatedconditions such as anti-Flaviviridae antibody positive andFlaviviridae-positive conditions, chronic liver inflammation caused byHCV, cirrhosis, acute hepatitis, fulminant hepatitis, chronic persistenthepatitis, and fatigue. These compounds or formulations can also be usedprophylactically to prevent or retard the progression of clinicalillness in individuals who are anti-Flaviviridae antibody orFlaviviridae-antigen positive or who have been exposed to aFlaviviridae. In one specific embodiment, the Flaviviridae is hepatitisC. In an alternative embodiment, the compound is used to treat any virusthat replicates through an RNA-dependent RNA polymerase.

A method for the treatment of a Flaviviridae infection in a host,including a human, is also disclosed that includes administering aneffective amount of a 2′- or 3′-prodrug of a biologically active 1′, 2′,3′ or 4′-branched β-D or β-L nucleosides or a pharmaceuticallyacceptable salt thereof, administered either alone or in combination oralternation with another anti-Flaviviridae agent, optionally in apharmaceutically acceptable carrier. The term 1′, 2′, 3′ or 4′-branched,as used in this specification, refers to a nucleoside that has anadditional non-natural substituent in the 1′, 2′, 3′ or 4′-position(i.e., carbon is bound to four nonhydrogen substituents). The term2′-prodrug, as used herein, refers to a 1′, 2′, 3′ or 4′-branched β-D orβ-L nucleoside that has a biologically cleavable moiety at the2′-position, including, but not limited to acyl, and in one embodiment,a natural or synthetic L- or D-amino acid, preferably an L-amino acid.The term 3′-prodrug, as used herein, refers to a 1′, 2′, 3′ or4′-branched β-D or β-L nucleoside that has a biologically cleavablemoiety at the 3′-position, including, but not limited to acyl, and inone embodiment, a natural or synthetic L- or D-amino acid, preferably anL-amino acid. Certain other alternative embodiments are also included.

In one embodiment, the prodrug of 1′, 2′, 3′ or 4′-branched β-D or β-Lnucleoside includes biologically cleavable moieties at the 2′ and/or 5′positions. Preferred moieties are natural or synthetic D or L amino acidesters, including D or L-valyl, though preferably L-amino acid esters,such as L-valyl, and alkyl esters including acetyl. Therefore, thisinvention specifically includes 2′-L or D-amino acid ester and 2′,5′-Lor D-diamino acid ester of 1′, 2′, 3′ or 4′-branched β-D or β-Lnucleosides, preferably L-amino acid, with any desired purine orpyrimidine base, wherein the parent drug optionally has an EC₅₀ of lessthan 15 micromolar, and even more preferably less than 10 micromolar;2′-(alkyl or aryl) ester or 2′,5′-L-di(alkyl or aryl) ester of 1′, 2′,3′ or 4′-branched β-D or β-L nucleosides with any desired purine orpyrimidine base, wherein the parent drug optionally has an EC₅₀ of lessthan 10 or 15 micromolar; and prodrugs of 2′,5′-diesters of 1′, 2′, 3′or 4′-branched β-D or β-L nucleosides wherein (i) the 2′ ester is anamino acid ester and the 5′-ester is an alkyl or aryl ester; (ii) bothesters are amino acid esters; (iii) both esters are independently alkylor aryl esters; and (iv) the 2′ ester is independently an alkyl or arylester and the 5′-ester is an amino acid ester, wherein the parent drugoptionally has an EC₅₀ of less than 10 or 15 micromolar.

Examples of prodrugs falling within the invention are 2′-L-valine esterof β-D-2′-methyl-cytidine; 2′-L-valine ester of β-D-2′-methyl-thymidine;2′-L-valine ester of β-D-2′-methyl-adenosine; 2′-L-valine ester ofβ-D-2′-methyl-guanosine; 2′-L-valine ester ofβ-D-2′-methyl-5-fluorocytidine; 2′-L-valine ester ofβ-D-2′-methyl-uridine; 2′-acetyl ester of β-D-2′-methyl-cytidine;2′-acetyl ester of β-D-2′-methyl-thymidine; 2′-acetyl ester ofβ-D-2′-methyl-adenosine; 2′-acetyl ester of β-D-2′-methyl-guanosine;2′-acetyl ester of β-D-2′-methyl-5-fluoro-cytidine; and 2′-esters ofβ-D-2′-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,adenosine, or thymidine) wherein (i) the 2′ ester is an amino acidester; or (ii) the 2′ ester is an alkyl or aryl ester.

Additional examples of prodrugs falling within the invention are2′,5′-L-divaline ester of β-D-2′-methyl-cytidine (dival-2′-Me-L-dC);2′,5′-L-divaline ester of β-D-2′-methyl-thymidine; 2′,5′-L-divalineester of β-D-2′-methyl-adenosine; 2′,5′-L-divaline ester ofβ-D-2′-methyl-guanosine; 2′,5′-L-divaline ester ofβ-D-2′-methyl-5-fluoro-cytidine; 2′,5′-L-divaline ester ofβ-D-2′-methyl-uridine; 2′,5′-diacetyl ester of β-D-2′-methyl-cytidine;2′,5′-diacetyl ester of β-D-2′-methyl-thymidine; 2′,5′-diacetyl ester ofβ-D-2′-methyl-adenosine; 2′,5′-diacetyl ester ofβ-D-2′-methyl-guanosine; 2′,5′-diacetyl ester ofβ-D-2′-methyl-5-fluoro-cytidine; and 2′,5′-diesters ofβ-D-2′-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,adenosine, or thymidine) wherein (i) the 2′ ester is an amino acid esterand the 5′-ester is an alkyl or aryl ester; (ii) both esters are aminoacid esters; (iii) both esters are independently alkyl or aryl esters;or (iv) the 2′ ester is an alkyl or aryl ester and the 5′-ester is anamino acid ester.

In another embodiment, the prodrug of 1′, 2′, 3′ or 4′-branched β-D orβ-L nucleoside includes biologically cleavable moieties at the 3′ and/or5′ positions. Preferred moieties are natural or synthetic D or L aminoacid esters, including D or L-valyl, though preferably L-amino acidesters, such as L-valyl, and alkyl esters including acetyl. Therefore,this invention specifically includes 3′-L or D-amino acid ester and3′,5′-L or D-diamino acid ester of 1′, 2′, 3′ or 4′-branched β-D or β-Lnucleosides, preferably L-amino acid, with any desired purine orpyrimidine base, wherein the parent drug optionally has an EC₅₀ of lessthan 15 micromolar, and even more preferably less than 10 micromolar;3′-(alkyl or aryl) ester or 3′,5′-L-di(alkyl or aryl) ester of 1′, 2′,3′ or 4′-branched β-D or β-L nucleosides with any desired purine orpyrimidine base, wherein the parent drug optionally has an EC₅₀ of lessthan 10 or 15 micromolar; and prodrugs of 3′,5′-diesters of 1′, 2′, 3′or 4′-branched β-D or β-L nucleosides wherein (i) the 3′ ester is anamino acid ester and the 5′-ester is an alkyl or aryl ester; (ii) bothesters are amino acid esters; (iii) both esters are independently alkylor aryl esters; and (iv) the 3′ ester is independently an alkyl or arylester and the 5′-ester is an amino acid ester, wherein the parent drugoptionally has an EC₅₀ of less than 10 or 15 micromolar.

Examples of prodrugs falling within the invention are 3′-L-valine esterof β-D-2′-methyl-cytidine; 3′-L-valine ester of β-D-2′-methyl-thymidine;3′-L-valine ester of β-D-2′-methyl-adenosine; 3′-L-valine ester ofβ-D-2′-methyl-guanosine; 3′-L-valine ester ofβ-D-2′-methyl-5-fluorocytidine; 3′-L-valine ester ofβ-D-2′-methyl-uridine; 3′-acetyl ester of β-D-2′-methyl-cytidine;3′-acetyl ester of β-D-2′-methyl-thymidine; 3′-acetyl ester ofβ-D-2′-methyl-adenosine; 3′-acetyl ester of β-D-2′-methyl-guanosine;3′-acetyl ester of β-D-2′-methyl-5-fluoro-cytidine; and 3′-esters ofβ-D-2′-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,adenosine, or thymidine) wherein (i) the 3′ ester is an amino acidester; or (ii) the 3′ ester is an alkyl or aryl ester.

Additional examples of prodrugs falling within the invention are3′,5′-L-divaline ester of β-D-2′-methyl-cytidine (dival-2′-Me-L-dC);3′,5′-L-divaline ester of β-D-2′-methyl-thymidine; 3′,5′-L-divalineester of β-D-2′-methyl-adenosine; 3′,5′-L-divaline ester ofβ-D-2′-methyl-guanosine; 3′,5′-L-divaline ester ofβ-D-2′-methyl-5-fluoro-cytidine; 3′,5′-L-divaline ester ofβ-D-2′-methyl-uridine; 3′,5′-diacetyl ester of β-D-2′-methyl-cytidine;3′,5′-diacetyl ester of β-D-2′-methyl-thymidine; 3′,5′-diacetyl ester ofβ-D-2′-methyl-adenosine; 3′,5′-diacetyl ester ofβ-D-2′-methyl-guanosine; 3′,5′-diacetyl ester ofβ-D-2′-methyl-5-fluoro-cytidine; and 3′,5′-diesters ofβ-D-2′-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,adenosine, or thymidine) wherein (i) the 3′ ester is an amino acid esterand the 5′-ester is an alkyl or aryl ester; (ii) both esters are aminoacid esters; (iii) both esters are independently alkyl or aryl esters;or (iv) the 3′ ester is an alkyl or aryl ester and the 5′-ester is anamino acid ester.

In another embodiment, the prodrug of 1′, 2′, 3′ or 4′-branched β-D orβ-L nucleoside includes biologically cleavable moieties at the 2′, 3′and/or 5′ positions. Preferred moieties are D or L amino acid esters,including D or L-valyl, though preferably L-amino acid esters, such asL-valyl, and alkyl esters including acetyl. Therefore, this inventionspecifically includes 2′,3′-L or D-diamino acid ester and 2′,3′,5′-L orD-triamino acid ester of 1′, 2′, 3′ or 4′-branched β-D or β-Lnucleosides, preferably L-amino acid, with any desired purine orpyrimidine base, wherein the parent drug optionally has an EC₅₀ of lessthan 15 micromolar, and even more preferably less than 10 micromolar;2′,3′-di(alkyl or aryl) ester or 2′,3′,5′-L-tri(alkyl or aryl) ester of1′, 2′, 3′ or 4′-branched β-D or β-L nucleosides with any desired purineor pyrimidine base, wherein the parent drug optionally has an EC₅₀ ofless than 10 or 15 micromolar; and prodrugs of 2′,3′-diesters of 1′, 2′,3′ or 4′-branched β-D or β-L nucleosides wherein (i) the 2′ ester is anamino acid ester and the 3′-ester is an alkyl or aryl ester; (ii) bothesters are amino acid esters; (iii) both esters are independently alkylor aryl esters; and (iv) the 2′ ester is independently an alkyl or arylester and the 3′-ester is an amino acid ester, wherein the parent drugoptionally has an EC₅₀ of less than 10 or 15 micromolar. Further,2′,3′,5′-triesters of 1′, 2′, 3′ or 4′-branched β-D or β-L nucleosideswherein (i) all three esters are amino acid esters; (ii) all threeesters are independently alkyl or aryl esters; (iii) the 2′ ester is anamino acid ester, the 3′ ester is an amino acid ester and the 5′-esteris an alkyl or aryl ester; (iv) the 2′ ester is an amino acid ester, the3′ ester is an alkyl or aryl ester and the 5′-ester is an alkyl or arylester; (v) the 2′ ester is an alkyl or aryl ester, the 3′ ester is analkyl or aryl ester and the 5′-ester is an amino acid ester; (vi) the 2′ester is an alkyl or aryl ester, the 3′ ester is an amino acid ester andthe 5′-ester is an amino acid ester; (vii) the 2′ ester is an alkyl oraryl ester, the 3′ ester is an amino acid ester and the 5′-ester is analkyl or aryl ester; and (viii) the 2′ ester is an amino acid ester, the3′ ester is an alkyl or aryl ester and the 5′-ester is an amino acidester; wherein the parent drug optionally has an EC₅₀ of less than 10 or15 micromolar.

Examples of prodrugs falling within the invention include2′,3′-L-divaline ester of β-D-2′-methyl-cytidine (dival-2′-Me-L-dC);2′,3′-L-divaline ester of β-D-2′-methyl-thymidine; 2′,3′-L-divalineester of β-D-2′-methyl-adenosine; 2′,3′-L-divaline ester ofβ-D-2′-methyl-guanosine; 2′,3′-L-divaline ester ofβ-D-2′-methyl-5-fluoro-cytidine; 2′,3′-L-divaline ester ofβ-D-2′-methyl-uridine; 2′,3′-diacetyl ester of β-D-2′-methyl-cytidine;2′,3′-diacetyl ester of β-D-2′-methyl-thymidine; 2′,3′-diacetyl ester ofβ-D-2′-methyl-adenosine; 2′,3′-diacetyl ester ofβ-D-2′-methyl-guanosine; 2′,3′-diacetyl ester ofβ-D-2′-methyl-5-fluoro-cytidine; and 2′,3′-diesters ofβ-D-2′-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,adenosine, or thymidine) wherein (i) the 2′ ester is an amino acid esterand the 3′-ester is an alkyl or aryl ester; (ii) both esters are aminoacid esters; (iii) both esters are independently alkyl or aryl esters;or (iv) the 2′ ester is an alkyl or aryl ester and the 3′-ester is anamino acid ester.

Additional examples of prodrugs falling within the invention include2′,3′,5′-L-trivaline ester of β-D-2′-methyl-cytidine(trival-2′-Me-L-dC); 2′,3′,5′-L-trivaline ester ofβ-D-2′-methyl-thymidine; 2′,3′,5′-L-trivaline ester ofβ-D-2′-methyl-adenosine; 2′,3′,5′-L-trivaline ester ofβ-D-2′-methyl-guanosine; 2′,3′,5′-L-trivaline ester ofβ-D-2′-methyl-5-fluoro-cytidine; 2′,3′,5′-L-trivaline ester ofβ-D-2′-methyl-uridine; 2′,3′,5′-triacetyl ester ofβ-D-2′-methyl-cytidine; 2′,3′,5′-triacetyl ester ofβ-D-2′-methyl-thymidine; 2′,3′,5′-triacetyl ester ofβ-D-2′-methyl-adenosine; 2′,3′,5′-triacetyl ester ofβ-D-2′-methyl-guanosine; 2′,3′,5′-triacetyl ester ofβ-D-2′-methyl-5-fluoro-cytidine; and 2′,3′,5′-triesters ofβ-D-2′-methyl-(cytidine, 5-fluorocytidine, guanosine, uridine,adenosine, or thymidine) wherein (i) all three esters are amino acidesters; (ii) all three esters are independently alkyl or aryl esters;(iii) the 2′ ester is an amino acid ester, the 3′ ester is an amino acidester and the 5′-ester is an alkyl or aryl ester; (iv) the 2′ ester isan amino acid ester, the 3′ ester is an alkyl or aryl ester and the5′-ester is an alkyl or aryl ester; (v) the 2′ ester is an alkyl or arylester, the 3′ ester is an alkyl or aryl ester and the 5′-ester is anamino acid ester; (vi) the 2′ ester is an alkyl or aryl ester, the 3′ester is an amino acid ester and the 5′-ester is an amino acid ester;(vii) the 2′ ester is an alkyl or aryl ester, the 3′ ester is an aminoacid ester and the 5′-ester is an alkyl or aryl ester; and (viii) the 2′ester is an amino acid ester, the 3′ ester is an alkyl or aryl ester andthe 5′-ester is an amino acid ester.

Pharmaceutically acceptable salts of tosylate, methanesulfonate,acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate, formate, fumarate, propionate,glycolate, lactate, pyruvate, oxalate, maleate, salicyate, sulfate,sulfonate, nitrate, bicarbonate, hydrobromate, hydrobromide,hydroiodide, carbonate, and phosphoric acid salts are provided. Aparticularly preferred embodiment is the mono or dihydrochloridepharmaceutically acceptable salts.

In a first principal embodiment, a compound of Formula (I), or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which is capable of providing a compoundwherein R¹, R² and/or R³ is independently H or phosphate, for examplewhen administered in vivo;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OH, OR⁴, NH, NHR⁵, NR⁴R⁵, SHand SR⁴;X¹ and X² are independently selected from the group consisting of H,straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OH, OR⁴, NH, NHR⁵, NR⁴R⁵,SH and SR⁴; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In the embodiments described herein, R¹, R² and/or R³ can independentlybe a pharmaceutically acceptable leaving group which is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate (including mono-, di- or triphosphate), for example whenadministered in vivo.

In a second principal embodiment, a compound of Formula II, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OR⁴, NR⁴R⁵ or SR⁴;X¹ and X² are independently selected from the group consisting of H,straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OR⁴, NR⁴NR⁵ or SR⁵; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a third principal embodiment, a compound of Formula III, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OR⁴, NR⁴R⁵ or SR⁴;

-   X¹ and X² are independently selected from the group consisting of H,    straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,    CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OR⁴, NR⁴NR⁵ or SR⁵; and    R⁴ and R⁵ are independently hydrogen, acyl (including lower acyl),    or alkyl (including but not limited to methyl, ethyl, propyl and    cyclopropyl).

In a fourth principal embodiment, a compound of Formula IV, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OR⁴, NR⁴R⁵ or SR⁴;X¹ is selected from the group consisting of H, straight chained,branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro,bromo, fluoro, iodo, OR⁴, NR⁴NR⁵ or SR⁵; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a fifth principal embodiment, a compound of Formula V, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OR⁴, NR⁴R⁵ or SR⁴;X¹ is selected from the group consisting of H, straight chained,branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro,bromo, fluoro, iodo, OR⁴, NR⁴NR⁵ or SR⁵; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a sixth principal embodiment, a compound of Formula VI, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OR⁴, NR⁴R⁵ or SR⁴;X¹ is selected from the group consisting of H, straight chained,branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro,bromo, fluoro, iodo, OR⁴, NR⁴NR⁵ or SR⁵; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a seventh principal embodiment, a compound selected from Formulas VIIand VIII,or a pharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:Base is a purine or pyrimidine base as defined herein;R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein. R¹, R² and/or R³ is independently H orphosphate;wherein R² is not hydrogen;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂; andX is O, S, SO₂ or CH₂.

In a eighth principal embodiment, a compound of Formulas IX and X, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:Base is a purine or pyrimidine base as defined herein;R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein R² is not hydrogen;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂; andR⁷ is hydrogen, OR³, hydroxy, alkyl (including lower alkyl), azido,cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl), —C(O)O(lower alkyl),—O(acyl), —O(lower acyl), —O(alkyl), —O(lower alkyl), —O(alkenyl),chlorine, bromine, iodine, NO₂, NH₂, —NH(lower alkyl), —NH(acyl),—N(lower alkyl)₂, —N(acyl)₂; andX is O, S, SO₂ or CH₂.

In a ninth principal embodiment a compound selected from Formulas XI andXII, or a pharmaceutically acceptable salt or prodrug thereof, isprovided:

wherein:Base is a purine or pyrimidine base as defined herein;R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein R² is not hydrogen;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂; andX is O, S, SO₂ or CH₂.

In a tenth principal embodiment the invention provides a compound ofFormula XIII,or a pharmaceutically acceptable salt or prodrug thereof:

wherein:Base is a purine or pyrimidine base as defined herein;R¹ is H, phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R¹ is H or phosphate;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;R⁷ and R⁹ are independently hydrogen, OR², hydroxy, alkyl (includinglower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), chlorine, bromine, iodine, NO₂, NH₂, —NH(loweralkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;wherein at least one of R⁷ and R⁹ is OR², wherein the R² isindependently phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R² is H or phosphate;R⁸ and R¹⁰ are independently H, alkyl (including lower alkyl), chlorine,bromine or iodine; alternatively, R⁷ and R¹⁰, R⁸ and R⁹, or R⁸ and R¹⁰can come together to form a pi bond; andX is O, S, SO₂ or CH₂.

In a eleventh principal embodiment the invention provides a compound ofFormula XIV, or a pharmaceutically acceptable salt or prodrug thereof:

wherein:Base is a purine or pyrimidine base as defined herein;R¹ is H, phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R¹ is H or phosphate;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;R⁷ and R⁹ are independently hydrogen, OR², hydroxy, alkyl (includinglower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), chlorine, bromine, iodine, NO₂, NH₂, —NH(loweralkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;wherein at least one of R⁷ and R⁹ is OR², wherein the R² isindependently phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R² is H or phosphate;R¹⁰ is H, alkyl (including lower alkyl), chlorine, bromine or iodine;alternatively, or R⁷ and R¹⁰ can come together to form a pi bond; andX is O, S, SO₂ or CH₂.

In a twelfth principal embodiment, the invention provides a compound ofFormula XV, or a pharmaceutically acceptable salt or prodrug thereof:

wherein:Base is a purine or pyrimidine base as defined herein;R¹ is H, phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R¹ is H or phosphate;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;R⁷ and R⁹ are independently hydrogen, OR², hydroxy, alkyl (includinglower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), chlorine, bromine, iodine, NO₂, NH₂, —NH(loweralkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;wherein at least one of R⁷ and R⁹ is OR², wherein each R² isindependently phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R² is H or phosphate;R⁹ is H, alkyl (including lower alkyl), chlorine, bromine or iodine;alternatively, R⁸ and R⁹ can come together to form a pi bond;X is O, S, SO₂ or CH₂.

In a thirteenth principal embodiment, a compound of Formula XVI, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OH, OR⁴, NH, NHR⁵, NR⁴R⁵, SHand SR⁴;X¹ and X² are independently selected from the group consisting of H,straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OH, OR⁴, NH, NHR⁵, NR⁴R⁵,SH and SR⁴; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a fourteenth principal embodiment, a compound of Formula XVII, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OR⁴, NR⁴R⁵ or SR⁴;X¹ is selected from the group consisting of H, straight chained,branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro,bromo, fluoro, iodo, OR⁴, NR⁴NR⁵ or SR⁵; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a fifteenth principal embodiment, a compound selected from FormulasXVIII and XIX, or a pharmaceutically acceptable salt or prodrug thereof,is provided:

wherein:Base is a purine or pyrimidine base as defined herein;R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein R² is not hydrogen;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂; andX is O, S, SO₂ or CH₂.

In a sixteenth principal embodiment the invention provides a compound ofFormula XX, or a pharmaceutically acceptable salt or prodrug thereof:

wherein:Base is a purine or pyrimidine base as defined herein;R¹ is H, phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R¹ is H or phosphate;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;R⁷ and R⁹ are independently hydrogen, OR², hydroxy, alkyl (includinglower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), chlorine, bromine, iodine, NO₂, NH₂, —NH(loweralkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;wherein at least one of R⁷ and R⁹ is OR², wherein each R² isindependently phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R² is H or phosphate;R⁸ and R¹⁰ are independently H, alkyl (including lower alkyl), chlorine,bromine or iodine;alternatively, R⁷ and R¹⁰, R⁸ and R⁹, or R⁸ and R¹⁰ can come together toform a pi bond; andX is O, S, SO₂ or CH₂.

In one embodiment, the amino acid residue is of the formulaC(O)C(R¹¹)(R¹²)(NR¹³R¹⁴), wherein

R¹¹ is the side chain of an amino acid and wherein, as in proline, R¹¹can optionally be attached to R¹³ to form a ring structure; oralternatively, R¹¹ is an alkyl, aryl, heteroaryl or heterocyclic moiety;

R¹² is hydrogen, alkyl (including lower alkyl) or aryl; and

R¹³ and R¹⁴ are independently hydrogen, acyl (including an acylderivative attached to R¹¹) or alkyl (including but not limited tomethyl, ethyl, propyl, and cyclopropyl).

In another preferred embodiment, at least one of R² and R³ is an aminoacid residue, and is preferably L-valinyl.

The β-D- and β-L-nucleosides of this invention may inhibit HCVpolymerase activity. Nucleosides can be screened for their ability toinhibit HCV polymerase activity in vitro according to screening methodsset forth more particularly herein. One can readily determine thespectrum of activity by evaluating the compound in the assays describedherein or with another confirmatory assay.

In one embodiment the efficacy of the anti-HCV compound is measuredaccording to the concentration of compound necessary to reduce theplaque number of the virus in vitro, according to methods set forth moreparticularly herein, by 50% (i.e. the compound's EC₅₀). In preferredembodiments the parent of the prodrug compound exhibits an EC₅₀ of lessthan 25, 15, 10, 5, or 1 micromolar. In one embodiment the efficacy ofthe anti-Flaviviridae compound is measured according to theconcentration of compound necessary to reduce the plaque number of thevirus in vitro, according to methods set forth more particularly herein,by 50% (i.e. the compound's EC₅₀). In preferred embodiments the compoundexhibits an EC₅₀ of less than 15 or 10 micromolar, when measuredaccording to the polymerase assay described in Ferrari et al., J.Virol., 73:1649-1654, 1999; Ishii et al., Hepatology, 29:1227-1235,1999; Lohmann et al., J. Biol. Chem., 274:10807-10815, 1999; orYamashita et al, J. Biol. Chem., 273:15479-15486, 1998.

In another embodiment, combination and/or alternation therapy areprovided. In combination therapy, an effective dosage of two or moreagents are administered together, whereas during alternation therapy aneffective dosage of each agent is administered serially. The dosageswill depend on absorption, inactivation, and excretion rates of the drugas well as other factors known to those of skill in the art. It is to benoted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens and schedules should beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions.

The invention also provides combinations of at least two of the hereindescribed prodrugs. The invention further provides at least one of thedescribed 2′ and 3′-prodrugs in combination or alternation with a secondnucleoside that exhibits activity against a Flaviviridae virus,including but not limited to a parent drug of any of the prodrugsdefined herein, i.e. β-D-2′-methyl-cytidine, β-D-2′-methyl-thymidine,β-D-2′-methyl-adenosine, β-D-2′-methyl-guanosine,β-D-2′-methyl-5-fluorocytidine and/or β-D-2′-methyl-uridine.Alternatively, the 2′ or 3′-prodrugs can be administered in combinationor alternation with other anti-Flaviviridae agent exhibits an EC₅₀ ofless than 10 or 15 micromolar, or their prodrugs or pharmaceuticallyacceptable salts.

Nonlimiting examples of antiviral agents that can be used in combinationwith the compounds disclosed herein include:

(1) an interferon and/or ribavirin; (2) Substrate-based NS3 proteaseinhibitors; (3) Non-substrate-based inhibitors; (4) Thiazolidinederivatives; (5) Thiazolidines and benzanilides; (6) Aphenan-threnequinone; (7) NS3 inhibitors; (8) HCV helicase inhibitors;(9) polymerase inhibitors, including RNA-dependent RNA-polymeraseinhibitors; (10) Antisense oligodeoxynucleotides (11) Inhibitors ofIRES-dependent translation; (12) Nuclease-resistant ribozymes; and (13)other compounds that exhibit activity against a flaviviridae. Theinvention further includes administering the prodrug in combination oralternation with an immune modulator or other pharmaceutically activemodifer of viral replication, including a biological material such as aprotein, peptide, oligonucleotide, or gamma globulin, including but notlimited to interfereon, interleukin, or an antisense oligonucleotides togenes which express or regulate Flaviviridae replication.

The compounds described herein have a number of enantiomericconfigurations, any of which can be used as desired. The parentnucleoside framework can exist as a β-D or β-L nucleoside. In apreferred embodiment, the compound is administered in a form that is atleast 90% of the β-D enantiomer. In another embodiment, the compound isat least 95% of the β-D enantiomer. Certain prodrug acyl esters,specifically including amino acid esters, also have enantiomeric forms.In alternative embodiments, the compounds are used as racemic mixturesor as any combination of β-D or β-L parent nucleoside and L or D aminoacid.

In an alternative embodiment, the parent nucleoside compounds of any ofthe 2′ or 3′-prodrugs (i.e., the nucleosides without the 2′ or 3′cleavable moieties) provided for the treatment of a Flaviviridae, and inparticular, an HCV infection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the structure of various non-limiting examples ofnucleosides of the present invention, as well as other knownnucleosides, in particular FIAU and ribavirin.

FIG. 2 provides a non-limiting example of the steps involved inesterification of the 1′, 2′, 3′ or 4′-branched β-D or β-L nucleoside toobtain a 2′-prodrug. The same general procedure can be used to obtainthe 3′-prodrug by selectively protecting the 2′ and 5′-hydroxyl groupsor protecting the 2′, 3′ and 5′-hydroxyl groups and selectivelydeprotecting the 3′-hydroxyl.

FIG. 3 provides a non-limiting example of the steps involved inesterification of the 1′, 2′, 3′ or 4′-branched β-D or β-L nucleoside toobtain a 3′-prodrug.

FIG. 4 provides a non-limiting example of the esterification of the 1′,2′, 3′ or 4′-branched β-D or β-L nucleoside to obtain a 2′,3′-prodrug.

FIG. 5 is an illustration of a process of synthesizing aβ-D-2′-C-methyl-ribofuransyl-cytidine or a 3′-O-L-valine ester thereof.

FIG. 6 is an illustration of another process of synthesizing aβ-D-2′-C-methyl-ribofuransyl-cytidine or a 3′-O-L-valine ester thereof.

FIG. 7 is a diagram of a process of synthesizing aβ-D-2′-C-methyl-2′-acetyl-ribofuransyl-cytidine-3′-O-L-valine ester.

FIG. 8 is a diagram of a process of synthesizing aβ-D-2′-C-methyl-2′-acetyl-ribofuransyl-cytidine-3′-O-L-proline ester.

FIG. 9 is a diagram of a process of synthesizing aβ-D-2′-C-methyl-2′-acetyl-ribofuransyl-cytidine-3′-O-L-alanine ester.

FIG. 10 is a diagram of a process of synthesizing aβ-D-2′-C-methyl-2′-(cyclohexanecarboxylate)-ribofuransyl-cytidine-3′-O-L-valine ester.

FIG. 11 is a graph showing the concentration of BVDB (Log₁₀ units/ml)over a concentration range of four test compounds and ribavirin as acontrol in a cell based assay using de novo BVDV infected MDBK cells.This graph shows the antiviral potency of these compounds.

FIG. 12 is a photocopy of a gel illustrating the site-specific chaintermination of in vitro RNA synthesis by β-D-2′-C-methyl-ribofuranosylcytidine triphosphate at specified guanine residues in RNA templates, asdescribed in Example 32.

FIG. 13 is a graph of the titer of bovine viral diarrhea virus (BVDV)over number of passages of BVDV infected MDBK cells, indicatingeradication of a persistent BVDV infection by prolonged treatment withβ-D-2′-C-methyl-ribofuranosyl cytidine (16 uM) as described in Example33. Arrows indicate points at which a portion of cells were withdrawnfrom drug treatment.

FIGS. 14 a and 14 b are graphs of the concentration of bovine viraldiarrhea virus (BVDV) in MDBK cells persistently infected with thevirus, as described in Example 34. These graphs indicate the synergybetween β-D-2′-C-methyl-ribofuranosyl cytidine and interferon alpha 2b(IntronA) in reducing the viral titer. FIG. 14 a is a graph of theeffect of β-D-2′-C-methyl-ribofuranosyl cytidine and IntronA on BVDV(strain NY-1) titers in persistently infected MDBK cells over time. FIG.14 b is a graph of the effect of β-D-2′-C-methyl-ribofuranosyl cytidinein combination with IntronA on BVDV (strain I-N-dIns) titers inpersistently-infected MDBK cells.

FIG. 15 a-d illustrate the results of experiments studying thedevelopment of resistance to β-D-2′-C-methyl-ribofuranosyl cytidinetreated MDBK cells, infected with bovine viral diarrhea virus (BVDV), asdescribed in Example 35. FIG. 15 a is a graph of a representativeexperiment showing the effect over twenty eight days ofβ-D-2′-C-methyl-ribofuranosyl cytidine or IntronA treatment on BVDV(strain I-N-dIns) titers in persistently infected MDBK cells. FIG. 15 bis a photocopy of a dish plated with infected MDBK cells thatillustrates the size of the foci formed by phenotypes of the wild-typeBVDV (strain I-N-dIns), versus the β-D-2′-C-methyl-ribofuranosylcytidine-resistant BVDV (I-N-dIns 107R), indicating that the resistantvirus formed much smaller foci than the wild-type, I-N-dIns strain. FIG.15 c is a graph of the titer of BVDV strains I-N-dIns or I-N-dIns-107Rover hours post-infection in infected MDBK cells. FIG. 15 d is a graphof the effect of Intron A on the BVDV viral titer yield in denovo-infected MDBK cells treated with IntronA.

FIG. 16 is a graph of the concentration of hepatitis C virus (Log₁₀) inindividual chimpanzees over days of treatment withβ-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine ester as describedin Example 36.

FIG. 17 is a graph of the concentration of hepatitis C virus inindividual chimpanzees over days of treatment withβ-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine ester as comparedto baseline, as described in Example 36.

FIG. 18 is a graph of percent of total β-D-2′-C-methyl-ribofuranosylcytidine-3′-O-L-valine ester remaining in samples over time afterincubation of the drug in human plasma at 4° C., 21° C., and 37° C., asdescribed in Example 37.

FIG. 19 a is a graph showing the relative levels of the di- andtri-phosphate derivatives of β-D-2′-C-methyl-ribofuranosyl cytidine andβ-D-2′-C-methyl-ribofuranosyl uridine (mUrd) after incubation of HepG2cells with 10 ∥M βD-2′-C-methyl-ribofuranosyl cytidine over time, asdescribed in Example 37. FIG. 19 b is a graph of the decay of thetri-phosphate derivative of β-D-2′-C-methyl-ribofuranosyl cytidine afterincubation of HepG2 cells with 10 μM β-D-2′-C-methyl-ribofuranosylcytidine over time. FIG. 19 c is a graph of the concentration of the di-and tri-phosphate derivatives of β-D-2′-C-methyl-ribofuranosyl cytidineand β-D-2′-C-methyl-ribofuranosyl uridine (mUrd) after incubation ofHepG2 cells with 10M β-D-2′-C-methyl-ribofuranosyl cytidine atincreasing concentrations of the drug (μM).

FIG. 20 is a graph of the concentration (ng/ml) ofβ-D-2′-C-methyl-ribofuranosyl cytidine in human serum afteradministration of β-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valineester to patients, as described in Example 40.

FIG. 21 is a graph of the median change of the titer of hepatitis Cvirus in human patients after administration ofβ-D-2′-C-methyl-ribofuranosyl cytidine-3′-O-L-valine ester, as describedin Example 40. The graph indicates change from baseline in Log₁₀ HCV RNAby patient visit.

FIG. 22 is a table of the EC₅₀ and CC₅₀ of representative compounds in aBVDV cell protection assay.

DETAILED DESCRIPTION OF THE INVENTION

The invention as disclosed herein is a compound, a method andcomposition for the treatment of a Flaviviridae infection in humans andother host animals. The method includes the administration of aneffective HCV or Flaviviridae treatment amount of a 2′- or 3′-prodrug ofa 1′, 2′, 3′ or 4′-branched β-D or β-L nucleoside as described herein ora pharmaceutically acceptable salt, derivative or prodrug thereof,optionally in a pharmaceutically acceptable carrier. The compound ofthis invention either possesses antiviral (i.e., anti-HCV) activity, oris metabolized to a compound that exhibits such activity.

The 2′- or 3′-prodrug of a 1′, 2′, 3′ or 4′-branched β-D or β-Lnucleoside are acyl derivates of a secondary or tertiary alcohol alphato a secondary or tertiary carbon. Due to the steric hindrance of theseprodrugs over the 5′-prodrugs, an acyl derivative of a primary alcohol,these prodrugs differently modulate the biological properties of themolecule in vivo. It has been discovered that the 2′- and 3′-prodrugs ofa 1′, 2′, 3′ or 4′-branched β-D or β-L nucleoside can provide a drugwith increased half-life and improved pharmacokinetic profile.

The 2′- and 3′-prodrugs in a preferred embodiment is a cleavable acylgroup, and most particularly, an amino acid moiety, prepared from anynaturally occurring or synthetic α, β γ or δ amino acid, including butis not limited to, glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan, proline, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,arginine and histidine. In a preferred embodiment, the amino acid is inthe L-configuration. Alternatively, the amino acid can be a derivativeof alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl, phenylalaninyl,tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl,tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl,argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl,β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl,β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl,β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl orβ-histidinyl. In one particular, embodiment, the moiety is a valineester. One particularly preferred compound is the 3′-valine ester of2′-methyl-ribo-cytidine.

The oral bio-availability of 1′, 2′, 3′ or 4′-branched β-D or β-Lnucleoside as the neutral base and the HCl salt is low in rodents andnon-human primates. It has been discovered that there is significantcompetition of 1′, 2′, 3′ or 4′-branched β-D or β-L nucleoside withother nucleosides or nucleoside analogs for absorption, or transport,from the gastrointestinal tract and competition of other nucleosides ornucleoside analogs for the absorption with 1′, 2′, 3′ or 4′-branched β-Dor β-L nucleoside. In order to improve oral bioavailability and reducethe potential for drug-drug interaction, 2′ and 3′-prodrugs of 1′, 2′,3′ or 4′-branched β-D or β-L nucleoside were obtained with higher oralbioavailability than the parent molecule and a reduced effect on thebioavailability of other nucleosides or nucleoside analogs used incombination.

The 2′, 3′, and/or 5′-mono, di or trivaline ester of a 1′, 2′, 3′ or4′-branched β-D or β-L nucleoside have higher oral bioavailability thanthe parent 1′, 2′, 3′ or 4′-branched β-D or β-L nucleoside and reducedinteraction with other nucleosides or nucleoside analogs when used incombination as compared to 1′, 2′, 3′ or 4′-branched β-D or β-Lnucleoside.

The 2′, 3′, and/or 5′-mono, di or trivaline ester of a 1′, 2′, 3′ or4′-branched β-D or β-L nucleoside can be converted to the parent 1′, 2′,3′ or 4′-branched β-D or β-L nucleoside through de-esterification in thegastrointestinal mucosa, blood or liver. The 2′, 3′, and/or 5′-mono, dior trivaline ester of a 1′, 2′, 3′ or 4′-branched β-D or β-L nucleosidecan be actively transported from the gastrointestinal lumen after oraldelivery into the bloodstream by an amino acid transporter function inthe mucosa of the gastrointestinal tract. This accounts for the increasein oral bioavailability compared to the parent 1′, 2′, 3′ or 4′-branchedβ-D or β-L nucleoside that is transported primarily by a nucleosidetransporter function. There is reduced competition for uptake of the 2′,3′, and/or 5′-mono, di or trivaline ester of 1′, 2′, 3′ or 4′-branchedβ-D or β-L nucleoside with other nucleosides or nucleoside analogs thatare transported by the nucleoside transporter function and not the aminoacid transporter function. As partial de-esterification of the di ortrivaline ester of 1′, 2′, 3′ or 4′-branched β-D or β-L nucleosideoccurs prior to complete absorption, the mono or divaline estercontinues to be absorbed using the amino acid transporter function.Therefore, the desired outcome of better absorption, or bioavailability,and reduced competition with other nucleosides or nucleoside analogs foruptake into the bloodstream can be maintained.

In summary, the present invention includes the following features:

-   (a) a 2′ and/or 3′-prodrug of a 1′, 2′, 3′ or 4′-branched β-D or β-L    nucleoside, as described herein, and pharmaceutically acceptable    salts and compositions thereof;-   (b) a 2′ and/or 3′-prodrug of a 1′, 2′, 3′ or 4′-branched β-D or β-L    nucleoside as described herein, and pharmaceutically acceptable    salts and compositions thereof for use in the treatment and/or    prophylaxis of a Flaviviridae infection, especially in individuals    diagnosed as having a Flaviviridae infection or being at risk of    becoming infected by hepatitis C;-   (c) a 2′ and/or 3′-prodrug of a 1′, 2′, 3′ or 4′-branched β-D or β-L    nucleoside, or their pharmaceutically acceptable salts and    compositions as described herein substantially in the absence of the    opposite enantiomers of the described nucleoside, or substantially    isolated from other chemical entities;-   (d) processes for the preparation of a 2′ and/or 3′-prodrug of a 1′,    2′, 3′ or 4′-branched β-D or β-L nucleoside, as described in more    detail below;-   (e) pharmaceutical formulations comprising a 2′ and/or 3′-prodrug of    a 1′, 2′, 3′ or 4′-branched β-D or β-L nucleoside or a    pharmaceutically acceptable salt thereof together with a    pharmaceutically acceptable carrier or diluent;-   (f) pharmaceutical formulations comprising a 2′ and/or 3′-prodrug of    a 1′, 2′, 3′ or 4′-branched β-D or β-L nucleoside or a    pharmaceutically acceptable salt thereof together with one or more    other effective anti-HCV agents, optionally in a pharmaceutically    acceptable carrier or diluent;-   (g) pharmaceutical formulations comprising a 2′ and/or 3′-prodrug of    a 1′, 2′, 3′ or 4′-branched β-D or β-L nucleoside or a    pharmaceutically acceptable salt thereof together with the parent of    a different a 1′, 2′, 3′ or 4′-branched β-D or β-L nucleoside,    optionally in a pharmaceutically acceptable carrier or diluent;-   (h) a method for the treatment and/or prophylaxis of a host infected    with Flaviviridae that includes the administration of an effective    amount of a 2′ and/or 3′-prodrug of a 1′, 2′, 3′ or 4′-branched β-D    or β-L nucleoside, its pharmaceutically acceptable salt or    composition;-   (i) a method for the treatment and/or prophylaxis of a host infected    with Flaviviridae that includes the administration of an effective    amount of a 2′ and/or 3′-prodrug of a 1′, 2′, 3′ or 4′-branched β-D    or β-L nucleoside, its pharmaceutically acceptable salt or    composition in combination and/or alternation with one or more    effective anti-HCV agent;-   (j) a method for the treatment and/or prophylaxis of a host infected    with Flaviviridae that includes the administration of an effective    amount of a 2′ and/or 3′-prodrug of a 1′, 2′, 3′ or 4′-branched β-D    or β-L nucleoside, or its pharmaceutically acceptable salt or    composition with the parent of a different a 1′, 2′, 3′ or    4′-branched β-D or β-L nucleoside;-   (k) a method for the treatment and/or prophylaxis of a host infected    with Flaviviridae that includes the administration of an effective    amount of a 2′ and/or 3′-prodrug of a β-D-2′-methyl-cytidine, or its    pharmaceutically acceptable salt or composition thereof;-   (l) a method for the treatment and/or prophylaxis of a host infected    with Flaviviridae that includes the administration of an effective    amount of the 3′,5′-divalyl or diacetyl ester of    β-D-2′-methyl-cytidine, or its pharmaceutically acceptable salt or    composition thereof;-   (m) use of a 2′ and/or 3′-prodrug of a 1′, 2′, 3′ or 4′-branched β-D    or β-L nucleoside, and pharmaceutically acceptable salts and    compositions thereof for the treatment and/or prophylaxis of a    Flaviviridae infection in a host;-   (n) use of a 2′ and/or 3′-prodrug of a 1′, 2′, 3′ or 4′-branched β-D    or β-L nucleoside, its pharmaceutically acceptable salt or    composition in combination and/or alternation with one or more    effective anti-HCV agent for the treatment and/or prophylaxis of a    Flaviviridae infection in a host;-   (o) use of a 2′ and/or 3′-prodrug of a 1′, 2′, 3′ or 4′-branched β-D    or β-L nucleoside, or its pharmaceutically acceptable salt or    composition with the parent of a different a 1′, 2′, 3′ or    4′-branched β-D or β-L nucleoside for the treatment and/or    prophylaxis of a Flaviviridae infection in a host;-   (p) use of a 2′ and/or 3′-prodrug of a β-D-2′-methyl-cytidine, or    its pharmaceutically acceptable salt or composition thereof for the    treatment and/or prophylaxis of a Flaviviridae infection in a host;-   (q) use of the 3′,5′-divalyl or diacetyl ester of    β-D-2′-methyl-cytidine, or its pharmaceutically acceptable salt or    composition thereof for the treatment and/or prophylaxis of a    Flaviviridae infection in a host;-   (r) use of a 2′ and/or 3′-prodrug of a 1′, 2′, 3′ or 4′-branched β-D    or β-L nucleoside, and pharmaceutically acceptable salts and    compositions thereof in the manufacture of a medicament for    treatment and/or prophylaxis of a Flaviviridae infection;-   (s) use of a 2′ and/or 3′-prodrug of a 1′, 2′, 3′ or 4′-branched β-D    or β-L nucleoside, its pharmaceutically acceptable salt or    composition in combination and/or alternation with one or more    effective anti-HCV agent in the manufacture of a medicament for the    treatment and/or prophylaxis of a Flaviviridae infection in a host;-   (t) use of a 2′ and/or 3′-prodrug of a 1′, 2′, 3′ or 4′-branched β-D    or β-L nucleoside, or its pharmaceutically acceptable salt or    composition with the parent of a different a 1′, 2′, 3′ or    4′-branched β-D or β-L nucleoside in the manufacture of a medicament    for the treatment and/or prophylaxis of a Flaviviridae infection in    a host;-   (u) use of a 2′ and/or 3′-prodrug of a β-D-2′-methyl-cytidine, or    its pharmaceutically acceptable salt or composition thereof in the    manufacture of a medicament for the treatment and/or prophylaxis of    a Flaviviridae infection in a host; and-   (v) use of the 3′,5′-divalyl or diacetyl ester of    β-D-2′-methyl-cytidine, or its pharmaceutically acceptable salt or    composition thereof in the manufacture of a medicament for the    treatment and/or prophylaxis of a Flaviviridae infection in a host.

Flaviviridae included within the scope of this invention are discussedgenerally in Fields Virology, Editors: Fields, B. N., Knipe, D. M., andHowley, P. M., Lippincott-Raven Publishers, Philadelphia, Pa., Chapter31, 1996. In a particular embodiment of the invention, the Flaviviridaeis HCV. In an alternate embodiment of the invention, the Flaviviridae isa flavivirus or pestivirus. Specific flaviviruses include, withoutlimitation: Absettarov, Alfuy, Apoi, Aroa, Bagaza, Banzi, Bouboui,Bussuquara, Cacipacore, Carey Island, Dakar bat, Dengue 1, Dengue 2,Dengue 3, Dengue 4, Edge Hill, Entebbe bat, Gadgets Gully, Hanzalova,Hypr, Ilheus, Israel turkey meningoencephalitis, Japanese encephalitis,Jugra, Jutiapa, Kadam, Karshi, Kedougou, Kokobera, Koutango, Kumlinge,Kunjin, Kyasanur Forest disease, Langat, Louping ill, Meaban, Modoc,Montana myotis leukoencephalitis, Murray valley encephalitis, Naranjal,Negishi, Ntaya, Omsk hemorrhagic fever, Phnom-Penh bat, Powassan, RioBravo, Rocio, Royal Farm, Russian spring-summer encephalitis, Saboya,St. Louis encephalitis, Sal Vieja, San Perlita, Saumarez Reef, Sepik,Sokuluk, Spondweni, Stratford, Tembusu, Tyuleniy, Uganda S, Usutu,Wesselsbron, West Nile, Yaounde, Yellow fever, and Zika.

Pestiviruses included within the scope of this invention are discussedgenerally in Fields Virology, Editors: Fields, B. N., Knipe, D. M., andHowley, P. M., Lippincott-Raven Publishers, Philadelphia, Pa., Chapter33, 1996. Specific pestiviruses include, without limitation: bovineviral diarrhea virus (“BVDV”), classical swine fever virus (“CSFV,” alsocalled hog cholera virus), and border disease virus (“BDV”).

I. Active Compounds

In a first principal embodiment, a compound of Formula (I), or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OH, OR⁴, NH, NHR¹, NR⁴R⁵, SHand SR⁴;X¹ and X² are independently selected from the group consisting of H,straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OH, OR⁴, NH, NHR⁵, NR⁴R⁵,SH and SR⁴; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a preferred subembodiment, a compound of Formula I, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

X¹ is H;

X² is H or NH₂; and

Y is hydrogen, bromo, chloro, fluoro, iodo, NH₂ or OH.

In a second principal embodiment, a compound of Formula II, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OR⁴, NR⁴R⁵ or SR⁴;X¹ and X² are independently selected from the group consisting of H,straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OR⁴, NR⁴NR⁵ or SR⁵; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a preferred subembodiment, a compound of Formula II, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

X¹ is H;

X² is H or NH₂; and

Y is hydrogen, bromo, chloro, fluoro, iodo, NH₂ or OH.

In a third principal embodiment, a compound of Formula III, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OR⁴, NR⁴R⁵ or SR⁴;X¹ and X² are independently selected from the group consisting of H,straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OR⁴, NR⁴NR⁵ or SR⁵; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a preferred subembodiment, a compound of Formula III, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

X¹ is H;

X² is H or NH₂; and

Y is hydrogen, bromo, chloro, fluoro, iodo, NH₂ or OH.

In a fourth principal embodiment, a compound of Formula IV, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OR⁴, NR⁴R⁵ or SR⁴;X¹ is selected from the group consisting of H, straight chained,branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro,bromo, fluoro, iodo, OR⁴, NR⁴NR⁵ or SR⁵; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a preferred subembodiment, a compound of Formula IV, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

X¹ is H or CH₃; and

Y is hydrogen, bromo, chloro, fluoro, iodo, NH₂ or OH.

In a fifth principal embodiment, a compound of Formula V, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OR⁴, NR⁴R⁵ or SR⁴;X¹ is selected from the group consisting of H, straight chained,branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro,bromo, fluoro, iodo, OR⁴, NR⁴NR⁵ or SR⁵; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a preferred subembodiment, a compound of Formula V, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

X¹ is H or CH₃; and

Y is hydrogen, bromo, chloro, fluoro, iodo, NH₂ or OH.

In a sixth principal embodiment, a compound of Formula VI, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OR⁴, NR⁴R⁵ or SR⁴;X¹ is selected from the group consisting of H, straight chained,branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro,bromo, fluoro, iodo, OR⁴, NR⁴NR⁵ or SR⁵; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a preferred subembodiment, a compound of Formula VI, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

X¹ is H or CH₃; and

Y is hydrogen, bromo, chloro, fluoro, iodo, NH₂ or OH.

In a seventh principal embodiment, a compound selected from Formulas VIIand VIII,or a pharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:Base is a purine or pyrimidine base as defined herein;R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein R² is not hydrogen;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂; andX is O, S, SO₂ or CH₂.

In a first subembodiment, a compound of Formula VII or VIII, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

Base is a purine or pyrimidine base as defined herein;

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

R⁶ is alkyl; and

X is OS, SO₂ or CH₂.

In a second subembodiment, a compound of Formula VII or VIII, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

Base is a purine or pyrimidine base as defined herein;

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is an amino acid residue;

R⁶ is alkyl; and

X is O, S, SO₂ or CH₂.

In a third subembodiment, a compound of Formula VII or VIII, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

Base is a purine or pyrimidine base as defined herein;

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

R⁶ is alkyl; and

X is O.

In a eighth principal embodiment, a compound of Formulas IX and X, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:Base is a purine or pyrimidine base as defined herein;R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein R² is not hydrogen;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂; andR⁷ is hydrogen, OR³, hydroxy, alkyl (including lower alkyl), azido,cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl), —C(O)O(lower alkyl),—O(acyl), —O(lower acyl), —O(alkyl), —O(lower alkyl), —O(alkenyl),chlorine, bromine, iodine, NO₂, NH₂, —NH(lower alkyl), —NH(acyl),—N(lower alkyl)₂, —N(acyl)₂; andX is O, S, SO₂ or CH₂.

In a first subembodiment, a compound of Formula IX or X, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

Base is a purine or pyrimidine base as defined herein;

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

R⁶ is alkyl; and

X is O, S, SO₂ or CH₂.

In a second subembodiment, a compound of Formula IX or X, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

Base is a purine or pyrimidine base as defined herein;

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is an amino acid residue;

R⁶ is alkyl; and

X is O, S, SO₂ or CH₂.

In a third subembodiment, a compound of Formula IX or X, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

Base is a purine or pyrimidine base as defined herein;

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

R⁶ is alkyl; and

X is O.

In another subembodiments, a compound of Formula X(a), or itspharmaceutically acceptable salt or prodrug, is provided:

wherein:Base is a purine or pyrimidine base as defined herein; optionallysubstituted with an amine or cyclopropyl (e.g., 2-amino, 2,6-diamino orcyclopropyl guanosine); andR¹ and R² are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein R² is not hydrogen.

In a ninth principal embodiment a compound selected from Formulas XI andXII, or a pharmaceutically acceptable salt or prodrug thereof, isprovided:

wherein:Base is a purine or pyrimidine base as defined herein;R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein R² is not hydrogen;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂; andX is O, S, SO₂ or CH₂.

In a first subembodiment, a compound of Formula XI or XII, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

Base is a purine or pyrimidine base as defined herein;

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

R⁶ is alkyl; and

X is O, S, SO₂ or CH₂.

In a second subembodiment, a compound of Formula XI or XII, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

Base is a purine or pyrimidine base as defined herein;

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is an amino acid residue;

R⁶ is alkyl; and

X is O, S, SO₂ or CH₂.

In a third subembodiment, a compound of Formula XI or XII, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

Base is a purine or pyrimidine base as defined herein;

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

R⁶ is alkyl; and

X is O.

In a tenth principal embodiment the invention provides a compound ofFormula XIII, or a pharmaceutically acceptable salt or prodrug thereof:

wherein:Base is a purine or pyrimidine base as defined herein; R¹ is H,phosphate (including mono-, di- or triphosphate and a stabilizedphosphate); straight chained, branched or cyclic alkyl (including loweralkyl); acyl (including lower acyl); CO-alkyl, CO-aryl, CO-alkoxyalkyl,CO-aryloxyalkyl, CO-substituted aryl, sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; alkylsulfonyl,arylsulfonyl, aralkylsulfonyl, a lipid, including a phospholipid; anamino acid; and amino acid residue, a carbohydrate; a peptide;cholesterol; or other pharmaceutically acceptable leaving group whichwhen administered in vivo is capable of providing a compound wherein R¹is H or phosphate;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;R⁷ and R⁹ are independently hydrogen, OR², hydroxy, alkyl (includinglower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), chlorine, bromine, iodine, NO₂, NH₂, —NH(loweralkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;wherein at least one of R⁷ and R⁹ is OR², wherein each R² isindependently phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R² is H or phosphate;R⁸ and R¹⁰ are independently H, alkyl (including lower alkyl), chlorine,bromine or iodine; alternatively, R⁷ and R¹⁰, R⁸ and R⁹, or R⁸ and R¹⁰can come together to form a pi bond; andX is O, S, SO₂ or CH₂.

In a first subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ are independently OR²,alkyl, alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine,NO₂, amino, lower alkylamino or di(loweralkyl)amino; wherein at leastone of R⁷ and R⁹ is OR² (and R² is not hydrogen); (5) R⁸ and R¹⁰ areindependently H, alkyl (including lower alkyl), chlorine, bromine, oriodine; and (6) X is O, S, SO₂ or CH₂.

In a second subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl, alkenyl, alkynyl, Br-vinyl, hydroxy,O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂, amino, loweralkylamino, or di(loweralkyl)amino; (4) R⁷ and R⁹ are independently OHor OR², wherein at least one of R⁷ and R⁹ is OR² (and R² is nothydrogen); (5) R⁸ and R¹⁰ are independently H, alkyl (including loweralkyl), chlorine, bromine, or iodine; and (6) X is O, S, SO₂ or CH₂.

In a third subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl, alkenyl, alkynyl, Br-vinyl, hydroxy,O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂, amino, loweralkylamino or di(loweralkyl)amino; (4) R⁷ and R⁹ are independently OR²,alkyl, alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine,NO₂, amino, lower alkylamino or di(loweralkyl)amino; wherein at leastone of R⁷ and R⁹ is OR² (and R² is not hydrogen); (5) R⁸ and R¹⁰ are H;and (6) X is O, S, SO₂ or CH₂.

In a fourth subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl, alkenyl, alkynyl, Br-vinyl, hydroxy,O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂, amino, loweralkylamino, or di(loweralkyl)amino; (4) R⁷ and R⁹ are independently OR²,alkyl, alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine,NO₂, amino, lower alkylamino, or di(loweralkyl)amino; wherein at leastone of R⁷ and R⁹ is OR² (and R² is not hydrogen); (5) R⁸ and R¹⁰ areindependently H, alkyl (including lower alkyl), chlorine, bromine, oriodine; and (6) X is O.

In a fifth subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ are independently OH orOR², wherein at least one of R⁷ and R⁹ is OR² (and R² is not hydrogen);(5) R⁸ and R¹⁰ are independently H, alkyl (including lower alkyl),chlorine, bromine or iodine; and (6) X is O, S, SO₂ or CH₂.

In a sixth subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ are independently OR²,alkyl (including lower alkyl), alkenyl, alkynyl, Br-vinyl, O-alkenyl,chlorine, bromine, iodine, NO₂, amino, lower alkylamino, ordi(loweralkyl)amino; wherein at least one of R⁷ and R⁹ is OR² (and R² isnot hydrogen); (5) R⁸ and R¹⁰ are H; and (6) X is O, S, SO₂, or CH₂.

In a seventh subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ are independently OR²,alkyl (including lower alkyl), alkenyl, alkynyl, Br-vinyl, O-alkenyl,chlorine, bromine, iodine, NO₂, amino, lower alkylamino ordi(loweralkyl)amino; wherein at least one of R⁷ and R⁹ is OR² (and R² isnot hydrogen); (5) R¹ and R¹⁰ are independently H, alkyl (includinglower alkyl), chlorine, bromine or iodine; and (6) X is O.

In a eighth subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R⁸ and R¹⁰ are hydrogen; and (6) X is O, S, SO₂or CH₂.

In a ninth subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R⁸ and R¹⁰ are independently H, alkyl(including lower alkyl), chlorine, bromine or iodine; and (6) X is O.

In a tenth preferred subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently OR², alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO₂, amino, loweralkylamino, or di(loweralkyl)amino; wherein at least one of R⁷ and R⁹ isOR² (and R² is not hydrogen); (5) R⁸ and R¹⁰ are hydrogen; and (6) X isO.

In an eleventh subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl (including lower alkyl),alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo,fluoro, iodo, NO₂, amino, lower alkylamino or di(loweralkyl)amino; (4)R⁷ and R⁹ are independently OH or OR², wherein at least one of R⁷ and R⁹is OR² (and R² is not hydrogen); (5) R⁸ and R¹⁰ are hydrogen; and (6) Xis O, S, SO₂ or CH₂.

In a twelfth subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R⁸ and R¹⁰ are hydrogen; and (6) X is O, S,SO₂, or CH₂.

In a thirteenth subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R⁸ and R¹⁰ are independently H, alkyl(including lower alkyl), chlorine, bromine, or iodine; and (6) X is O.

In a fourteenth subembodiment, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ areindependently OR², alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO₂, amino, loweralkylamino or di(loweralkyl)amino; wherein at least one of R⁷ and R⁹ isOR² (and R² is not hydrogen); (5) R⁸ and R¹⁰ are hydrogen; and (6) X isO.

In other subembodiments, a compound of Formula XIII, or itspharmaceutically acceptable salt or prodrug, is provided in which:

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is guanine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is cytosine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is thymidine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is uracil; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is adenine; (2) R¹ is phosphate; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is ethyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is propyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is butyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydrogen (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R³ and R¹⁰ are hydrogen; and (7) X isS;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isSO₂;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isCH₂.

In a eleventh principal embodiment the invention provides a compound ofFormula XIV, or a pharmaceutically acceptable salt or prodrug thereof:

wherein:

Base is a purine or pyrimidine base as defined herein;

R¹ is H, phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R¹ is H or phosphate;

R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;

R⁷ and R⁹ are independently hydrogen, OR², hydroxy, alkyl (includinglower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), chlorine, bromine, iodine, NO₂, NH₂, —NH(loweralkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;

wherein at least one of R⁷ and R⁹ is OR², wherein each R² isindependently phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R² is H or phosphate;

R¹⁰ is H, alkyl (including lower alkyl), chlorine, bromine or iodine;alternatively, or R⁷ and R¹⁰ can come together to form a pi bond; and

X is O, S, SO₂ or CH₂.

In a first subembodiment, a compound of Formula XIV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino, or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently hydrogen, OR², alkyl (including lower alkyl), alkenyl,alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO₂, amino,lower alkylamino or di(loweralkyl)-amino; wherein at least one of R⁷ andR⁹ is OR² (and R² is not hydrogen); (5) R¹⁰ is H; and (6) X is O, S,SO₂, or CH₂.

In a second subembodiment, a compound of Formula XIV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R¹⁰ is H, alkyl (including lower alkyl),chlorine, bromine, or iodine; and (6) X is O, S, SO₂ or CH₂.

In a third subembodiment, a compound of Formula XIV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino, or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently hydrogen, OR², alkyl (including lower alkyl), alkenyl,alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO₂, amino,lower alkylamino or di(loweralkyl)-amino; wherein at least one of R⁷ andR⁹ is OR² (and R² is not hydrogen); (5) R¹⁰ is H, alkyl (including loweralkyl), chlorine, bromine or iodine; and (6) X is O.

In a fourth subembodiment, a compound of Formula XIV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R¹⁰ is H; and (6) X is O, S, SO₂ or CH₂.

In a fifth subembodiment, a compound of Formula XIV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R¹⁰ is H, alkyl (including lower alkyl),chlorine, bromine or iodine; and (6) X is O.

In a sixth subembodiment, a compound of Formula XIV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino, or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently hydrogen, OR², alkyl (including lower alkyl), alkenyl,alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO₂, amino,lower alkylamino, or di(loweralkyl)amino; wherein at least one of R⁷ andR⁹ is OR² (and R² is not hydrogen); (5) R¹⁰ is H; and (6) X is O.

In a seventh subembodiment, a compound of Formula XIV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino, or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R¹⁰ is H; and (6) X is O.

In an eighth subembodiment, a compound of Formula XIV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ areindependently hydrogen, OR², alkyl (including lower alkyl), alkenyl,alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO₂, amino,lower alkylamino or di(loweralkyl)-amino; wherein at least one of R⁷ andR⁹ is OR² (and R² is not hydrogen); (5) R¹⁰ is H, alkyl (including loweralkyl), chlorine, bromine or iodine; and (6) X is O, S, SO₂, or CH₂.

In a ninth subembodiment, a compound of Formula XIV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl (including lower alkyl),alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo,fluoro, iodo, NO₂, amino, lower alkylamino, or di(loweralkyl)amino; (4)R⁷ and R⁹ are independently OH or OR², wherein at least one of R⁷ and R⁹is OR² (and R² is not hydrogen); (5) R¹⁰ is H; and (6) X is O, S, SO₂,or CH₂.

In a tenth preferred subembodiment, a compound of Formula XIV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R¹⁰ is H; and (6) X is O, S, SO₂, or CH₂.

In even more preferred subembodiments, a compound of Formula XIV, or itspharmaceutically acceptable salt or prodrug, is provided in which:

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R¹⁰ is hydrogen; and (7) X is O;

(1) Base is guanine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R¹⁰ is hydrogen; and (7) X is O;

(1) Base is cytosine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R¹⁰ is hydrogen; and (7) X is O;

(1) Base is thymidine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R¹⁰ is hydrogen; and (7) X is O;

(1) Base is uracil; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R¹⁰ is hydrogen; and (7) X is O;

(1) Base is adenine; (2) R¹ is phosphate; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R¹⁰ is hydrogen; and (7) X is O;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is ethyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R¹⁰ is hydrogen; and (7) X is O;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is propyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R¹⁰ is hydrogen; and (7) X is O;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is butyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R¹⁰ is hydrogen; and (7) X is O;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R¹⁰ is hydrogen; and (7) X is S;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R¹⁰ is hydrogen; and (7) X is SO₂; or

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R¹⁰ is hydrogen; and (7) X is CH₂.

In an twelfth principal embodiment, the invention provides a compound ofFormula XV, or a pharmaceutically acceptable salt or prodrug thereof:

wherein:Base is a purine or pyrimidine base as defined herein;R¹ is H, phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R¹ is H or phosphate;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;R⁷ and R⁹ are independently hydrogen, OR², hydroxy, alkyl (includinglower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), chlorine, bromine, iodine, NO₂, NH₂, —NH(loweralkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;wherein at least one of R⁷ and R⁹ is OR², wherein each R² isindependently phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R² is H or phosphate;R⁸ is H, alkyl (including lower alkyl), chlorine, bromine or iodine;alternatively, R⁸ and R⁹ can come together to form a pi bond;X is O, S, SO₂ or CH₂.

In a first subembodiment, a compound of Formula XV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including, lower alkyl); sulfonate ester including alkylor arylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ are independently hydrogen,OR², alkyl (including lower alkyl), alkenyl, alkynyl, Br-vinyl,O-alkenyl, chlorine, bromine, iodine, NO₂, amino, lower alkylamino ordi(loweralkyl)amino; wherein at least one of R⁷ and R⁹ is OR² (and R² isnot hydrogen); (5) R⁸ is H, alkyl (including lower alkyl), chlorine,bromine or iodine; and (6) X is O, S, SO₂ or CH₂.

In a second subembodiment, a compound of Formula XV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino or di-(loweralkyl)amino; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R⁸ is H, alkyl (including lower alkyl),chlorine, bromine, or iodine; and (6) X is O, S, SO₂ or CH₂.

In a third subembodiment, a compound of Formula XV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino, or di(lower-alkyl)amino; (4) R⁷ and R⁹ areindependently hydrogen, OR², alkyl (including lower alkyl), alkenyl,alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO₂, amino,lower alkylamino, or di(loweralkyl)amino; wherein at least one of R⁷ andR⁹ is OR² (and R² is not hydrogen); (5) R⁸ is H; and (6) X is O, S, SO₂or CH₂.

In a fourth subembodiment, a compound of Formula XV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino, or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently hydrogen, OR², alkyl (including lower alkyl), alkenyl,alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO₂, amino,lower alkylamino, or di(loweralkyl)amino; wherein at least one of R⁷ andR⁹ is OR² (and R² is not hydrogen); (5) R⁸ is H, alkyl (including loweralkyl), chlorine, bromine, or iodine; and (6) X is O.

In a fifth subembodiment, a compound of Formula XV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino, or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R⁸ is H; and (6) X is O, S, SO₂, or CH₂.

In a sixth subembodiment, a compound of Formula XV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino, or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R⁸ is H, alkyl (including lower alkyl),chlorine, bromine, or iodine; and (6) X is O.

In a seventh subembodiment, a compound of Formula XV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H; phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino, or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently hydrogen, OR², alkyl (including lower alkyl), alkenyl,alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO₂, amino,lower alkylamino, or di(loweralkyl)amino; wherein at least one of R⁷ andR⁹ is OR² (and R² is not hydrogen); (5) R⁸ is H; and (6) X is O.

In an eighth subembodiment, a compound of Formula XV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl (including lower alkyl),alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo,fluoro, iodo, NO₂, amino, lower alkylamino or di(loweralkyl)amino; (4)R⁷ and R⁹ are independently OH or OR², wherein at least one of R⁷ and R⁹is OR² (and R² is not hydrogen); (5) R⁸ is H; and (6) X is O, S, SO₂ orCH₂.

In a ninth subembodiment, a compound of Formula XV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R⁸ is H; and (6) X is O, S, SO₂, or CH₂.

In a tenth preferred subembodiment, a compound of Formula XV, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R⁸ is H; and (6) X is O.

In even more preferred subembodiments, a compound of Formula XV, or itspharmaceutically acceptable salt or prodrug, is provided in which:

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ is hydrogen; and (7) X is O;

(1) Base is guanine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ is hydrogen; and (7) X is O;

(1) Base is cytosine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ is hydrogen; and (7) X is O;

(1) Base is thymidine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ is hydrogen; and (7) X is O;

(1) Base is uracil; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ is hydrogen; and (7) X is O;

(1) Base is adenine; (2) R¹ is phosphate; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ is hydrogen; and (7) X is O;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is ethyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ is hydrogen; and (7) X is O;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is propyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ is hydrogen; and (7) X is O;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is butyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ is hydrogen; and (7) X is O;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ is hydrogen; and (7) X is S;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ is hydrogen; and (7) X is SO₂; or

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ is hydrogen; and (7) X is CH₂.

In a thirteenth principal embodiment, a compound of Formula XVI, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OH, OR⁴, NH, NHR⁵, NR⁴R⁵, SHand SR⁴;X¹ and X² are independently selected from the group consisting of H,straight chained, branched or cyclic alkyl, CO-alkyl, CO-aryl,CO-alkoxyalkyl, chloro, bromo, fluoro, iodo, OH, OR⁴, NH, NHR¹, NR⁴R⁵,SH and SR⁴; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a preferred subembodiment, a compound of Formula XVI, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

X¹ is H;

X² is H or NH₂; and

Y is hydrogen, bromo, chloro, fluoro, iodo, NH₂ or OH.

In a fourteenth principal embodiment, a compound of Formula XVII, or apharmaceutically acceptable salt or prodrug thereof, is provided:

wherein:R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein at least one of R² and R³ is not hydrogen;Y is hydrogen, bromo, chloro, fluoro, iodo, OR⁴, NR⁴R⁵ or SR⁴;X¹ is selected from the group consisting of H, straight chained,branched or cyclic alkyl, CO-alkyl, CO-aryl, CO-alkoxyalkyl, chloro,bromo, fluoro, iodo, OR⁴, NR⁴NR⁵ or SR⁵; andR⁴ and R⁵ are independently hydrogen, acyl (including lower acyl), oralkyl (including but not limited to methyl, ethyl, propyl andcyclopropyl).

In a preferred subembodiment, a compound of Formula XVII, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

X¹ is H or CH₃; and

Y is hydrogen, bromo, chloro, fluoro, iodo, NH₂ or OH.

In a fifteenth principal embodiment, a compound selected from FormulasXVIII and XIX, or a pharmaceutically acceptable salt or prodrug thereof,is provided:

wherein:Base is a purine or pyrimidine base as defined herein;R¹, R² and R³ are independently H, phosphate (including mono-, di- ortriphosphate and a stabilized phosphate); straight chained, branched orcyclic alkyl (including lower alkyl); acyl (including lower acyl);CO-alkyl, CO-aryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonate ester including alkyl or arylalkyl sulfonyl includingmethanesulfonyl and benzyl, wherein the phenyl group is optionallysubstituted with one or more substituents as described in the definitionof aryl given herein; alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, alipid, including a phospholipid; an amino acid; and amino acid residue,a carbohydrate; a peptide; cholesterol; or other pharmaceuticallyacceptable leaving group which when administered in vivo is capable ofproviding a compound wherein R¹, R² and/or R³ is independently H orphosphate;wherein R² is not hydrogen;R⁶ is alkyl (including lower alkyl and halogenated alkyl), CH₃, CF₃,azido, cyano, alkenyl, alkynyl, Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), CF₃, chloro, bromo, fluoro, iodo, NO₂, NH₂,—NH(lower alkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂; andX is O, S, SO₂ or CH₂.

In a first subembodiment, a compound of Formula XVIII and XIX, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

Base is a purine or pyrimidine base as defined herein;

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

R⁶ is alkyl; and

X is O, S, SO₂ or CH₂.

In a second subembodiment, a compound of Formula XVIII and XIX, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

Base is a purine or pyrimidine base as defined herein;

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is an amino acid residue;

R⁶ is alkyl; and

X is O, S, SO₂ or CH₂.

In a third subembodiment, a compound of Formula XVIII and XIX, or apharmaceutically acceptable salt or prodrug thereof, is providedwherein:

Base is a purine or pyrimidine base as defined herein;

R¹ is H or phosphate (preferably H);

R² and R³ are independently H, phosphate, acyl or an amino acid residue,wherein at least one of R² and R³ is acyl or an amino acid residue;

R⁶ is alkyl; and

X is O.

In a sixteenth principal embodiment the invention provides a compound ofFormula XX, or a pharmaceutically acceptable salt or prodrug thereof:

wherein:Base is a purine or pyrimidine base as defined herein;R¹ is H, phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R¹ is H or phosphate;R⁶ is alkyl (including lower alkyl), azido, cyano, alkenyl, alkynyl,Br-vinyl, 2-Br-ethyl, —C(O)O(alkyl), —C(O)O(lower alkyl), —O(acyl),—O(lower acyl), —O(alkyl), —O(lower alkyl), —O(alkenyl), CF₃, chloro,bromo, fluoro, iodo, NO₂, NH₂, —NH(lower alkyl), —NH(acyl), —N(loweralkyl)₂, —N(acyl)₂;R⁷ and R⁹ are independently hydrogen, OR², hydroxy, alkyl (includinglower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), chlorine, bromine, iodine, NO₂, NH₂, —NH(loweralkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;wherein at least one of R⁷ and R⁹ is OR², wherein each R² isindependently phosphate (including mono-, di- or triphosphate and astabilized phosphate); straight chained, branched or cyclic alkyl(including lower alkyl); acyl (including lower acyl); CO-alkyl, CO-aryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein;alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, a lipid, including aphospholipid; an amino acid; and amino acid residue, a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R² is H or phosphate;R⁸ and R¹⁰ are independently H, alkyl (including lower alkyl), chlorine,bromine or iodine; alternatively, R⁷ and R¹⁰, R⁸ and R⁹, or R⁸ and R¹⁰can come together to form a pi bond; andX is O, S, SO₂ or CH₂.

In a first subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ are independently OR²,alkyl, alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine,NO₂, amino, lower alkylamino or di(loweralkyl)amino; wherein at leastone of R⁷ and R⁹ is OR² (and R² is not hydrogen); (5) R⁸ and R¹⁰ areindependently H, alkyl (including lower alkyl), chlorine, bromine, oriodine; and (6) X is O, S, SO₂ or CH₂.

In a second subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl, alkenyl, alkynyl, Br-vinyl, hydroxy,O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂, amino, loweralkylamino, or di(loweralkyl)amino; (4) R⁷ and R⁹ are independently OHor OR², wherein at least one of R⁷ and R⁹ is OR² (and R² is nothydrogen); (5) R¹ and R¹⁰ are independently H, alkyl (including loweralkyl), chlorine, bromine, or iodine; and (6) X is O, S, SO₂ or CH₂.

In a third subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl, alkenyl, alkynyl, Br-vinyl, hydroxy,O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂, amino, loweralkylamino or di(loweralkyl)amino; (4) R⁷ and R⁹ are independently OR²,alkyl, alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine,NO₂, amino, lower alkylamino or di(loweralkyl)amino; wherein at leastone of R⁷ and R⁹ is OR² (and R² is not hydrogen); (5) R⁸ and R¹⁰ are H;and (6) X is O, S, SO₂ or CH₂.

In a fourth subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl, alkenyl, alkynyl, Br-vinyl, hydroxy,O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂, amino, loweralkylamino, or di(loweralkyl)amino; (4) R⁷ and R⁹ are independently OR²,alkyl, alkenyl, alkynyl, Br-vinyl, O-alkenyl, chlorine, bromine, iodine,NO₂, amino, lower alkylamino, or di(loweralkyl)amino; wherein at leastone of R⁷ and R⁹ is OR² (and R² is not hydrogen); (5) R⁸ and R¹⁰ areindependently H, alkyl (including lower alkyl), chlorine, bromine, oriodine; and (6) X is O.

In a fifth subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ are independently OH orOR², wherein at least one of R⁷ and R⁹ is OR² (and R² is not hydrogen);(5) R⁸ and R¹⁰ are independently H, alkyl (including lower alkyl),chlorine, bromine or iodine; and (6) X is O, S, SO₂ or CH₂.

In a sixth subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ are independently OR²,alkyl (including lower alkyl), alkenyl, alkynyl, Br-vinyl, O-alkenyl,chlorine, bromine, iodine, NO₂, amino, lower alkylamino, ordi(loweralkyl)amino; wherein at least one of R⁷ and R⁹ is OR² (and R² isnot hydrogen); (5) R⁸ and R¹⁰ are H; and (6) X is O, S, SO₂, or CH₂.

In a seventh subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ are independently OR²,alkyl (including lower alkyl), alkenyl, alkynyl, Br-vinyl, O-alkenyl,chlorine, bromine, iodine, NO₂, amino, lower alkylamino ordi(loweralkyl)amino; wherein at least one of R⁷ and R⁹ is OR² (and R² isnot hydrogen); (5) R⁸ and R¹⁰ are independently H, alkyl (includinglower alkyl), chlorine, bromine or iodine; and (6) X is O.

In a eighth subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R⁸ and R¹⁰ are hydrogen; and (6) X is O, S, SO₂or CH₂.

In a ninth subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R⁸ and R¹⁰ are independently H, alkyl(including lower alkyl), chlorine, bromine or iodine; and (6) X is O.

In a tenth preferred subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate (including monophosphate, diphosphate,triphosphate, or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); sulfonate ester including alkyl orarylalkyl sulfonyl including methanesulfonyl and benzyl, wherein thephenyl group is optionally substituted with one or more substituents asdescribed in the definition of aryl given herein; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; cholesterol; orother pharmaceutically acceptable leaving group which when administeredin vivo is capable of providing a compound wherein R¹ is independently Hor phosphate; (3) R⁶ is alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo, fluoro, iodo, NO₂,amino, lower alkylamino or di(loweralkyl)amino; (4) R⁷ and R⁹ areindependently OR², alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO₂, amino, loweralkylamino, or di(loweralkyl)amino; wherein at least one of R⁷ and R⁹ isOR² (and R² is not hydrogen); (5) R¹ and R¹⁰ are hydrogen; and (6) X isO.

In an eleventh subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl (including lower alkyl),alkenyl, alkynyl, Br-vinyl, hydroxy, O-alkyl, O-alkenyl, chloro, bromo,fluoro, iodo, NO₂, amino, lower alkylamino or di(loweralkyl)amino; (4)R⁷ and R⁹ are independently OH or OR², wherein at least one of R⁷ and R⁹is OR² (and R² is not hydrogen); (5) R⁸ and R¹⁰ are hydrogen; and (6) Xis O, S, SO₂ or CH₂.

In a twelfth subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R⁸ and R¹⁰ are hydrogen; and (6) X is O, S,SO₂, or CH₂.

In a thirteenth subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ areindependently OH or OR², wherein at least one of R⁷ and R⁹ is OR² (andR² is not hydrogen); (5) R⁸ and R¹⁰ are independently H, alkyl(including lower alkyl), chlorine, bromine, or iodine; and (6) X is O.

In a fourteenth subembodiment, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which: (1)Base is a purine or pyrimidine base as defined herein; (2) R¹ isindependently H or phosphate; (3) R⁶ is alkyl; (4) R⁷ and R⁹ areindependently OR², alkyl (including lower alkyl), alkenyl, alkynyl,Br-vinyl, O-alkenyl, chlorine, bromine, iodine, NO₂, amino, loweralkylamino or di(loweralkyl)amino; wherein at least one of R⁷ and R⁹ isOR² (and R² is not hydrogen); (5) R⁸ and R¹⁰ are hydrogen; and (6) X isO.

In even more preferred subembodiments, a compound of Formula XX, or itspharmaceutically acceptable salt or prodrug, is provided in which:

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is guanine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is cytosine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is thymidine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is uracil; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is adenine; (2) R¹ is phosphate; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is ethyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is propyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is butyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydrogen (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isO;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isS;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁸ and R¹⁰ are hydrogen; and (7) X isSO₂;

(1) Base is adenine; (2) R¹ is hydrogen; (3) R⁶ is methyl; (4) R⁷ ishydroxyl (5) R⁹ is L-valinyl; (6) R⁹ and R¹⁰ are hydrogen; and (7) X isCH₂.

Stereochemistry

It is appreciated that nucleosides of the present invention have severalchiral centers and may exist in and be isolated in optically active andracemic forms. Some compounds may exhibit polymorphism. It is to beunderstood that the present invention encompasses any racemic,optically-active, diastereomeric, polymorphic, or stereoisomeric form,or mixtures thereof, of a compound of the invention, which possess theuseful properties described herein. It being well known in the art howto prepare optically active forms (for example, by resolution of theracemic form by recrystallization techniques, by synthesis fromoptically-active starting materials, by chiral synthesis, or bychromatographic separation using a chiral stationary phase).

In particular, since the 1′ and 4′ carbons of the nucleoside are chiral,their nonhydrogen substituents (the base and the CHOR groups,respectively) can be either cis (on the same side) or trans (on oppositesides) with respect to the sugar ring system. The four optical isomerstherefore are represented by the following configurations (whenorienting the sugar moiety in a horizontal plane such that the oxygenatom is in the back): cis (with both groups “up”, which corresponds tothe configuration of naturally occurring β-D nucleosides), cis (withboth groups “down”, which is a normaturally occurring β-Lconfiguration), trans (with the C2′ substituent “up” and the C4′substituent “down”), and trans (with the C2′ substituent “down” and theC4′ substituent “up”). The “D-nucleosides” are cis nucleosides in anatural configuration and the “L-nucleosides” are cis nucleosides in thenon-naturally occurring configuration.

Likewise, most amino acids are chiral (designated as L or D, wherein theL enantiomer is the naturally occurring configuration) and can exist asseparate enantiomers.

Examples of methods to obtain optically active materials are known inthe art, and include at least the following.

-   -   i) physical separation of crystals—a technique whereby        macroscopic crystals of the individual enantiomers are manually        separated. This technique can be used if crystals of the        separate enantiomers exist, i.e., the material is a        conglomerate, and the crystals are visually distinct;    -   ii) simultaneous crystallization—a technique whereby the        individual enantiomers are separately crystallized from a        solution of the racemate, possible only if the latter is a        conglomerate in the solid state;    -   iii) enzymatic resolutions—a technique whereby partial or        complete separation of a racemate by virtue of differing rates        of reaction for the enantiomers with an enzyme;    -   iv) enzymatic asymmetric synthesis—a synthetic technique whereby        at least one step of the synthesis uses an enzymatic reaction to        obtain an enantiomerically pure or enriched synthetic precursor        of the desired enantiomer;    -   v) chemical asymmetric synthesis—a synthetic technique whereby        the desired enantiomer is synthesized from an achiral precursor        under conditions that produce asymmetry (i.e., chirality) in the        product, which may be achieved using chiral catalysts or chiral        auxiliaries;    -   vi) diastereomer separations—a technique whereby a racemic        compound is reacted with an enantiomerically pure reagent (the        chiral auxiliary) that converts the individual enantiomers to        diastereomers. The resulting diastereomers are then separated by        chromatography or crystallization by virtue of their now more        distinct structural differences and the chiral auxiliary later        removed to obtain the desired enantiomer;    -   vii) first- and second-order asymmetric transformations—a        technique whereby diastereomers from the racemate equilibrate to        yield a preponderance in solution of the diastereomer from the        desired enantiomer or where preferential crystallization of the        diastereomer from the desired enantiomer perturbs the        equilibrium such that eventually in principle all the material        is converted to the crystalline diastereomer from the desired        enantiomer. The desired enantiomer is then released from the        diastereomer;    -   viii) kinetic resolutions—this technique refers to the        achievement of partial or complete resolution of a racemate (or        of a further resolution of a partially resolved compound) by        virtue of unequal reaction rates of the enantiomers with a        chiral, non-racemic reagent or catalyst under kinetic        conditions;    -   ix) enantiospecific synthesis from non-racemic precursors—a        synthetic technique whereby the desired enantiomer is obtained        from non-chiral starting materials and where the stereochemical        integrity is not or is only minimally compromised over the        course of the synthesis;    -   x) chiral liquid chromatography—a technique whereby the        enantiomers of a racemate are separated in a liquid mobile phase        by virtue of their differing interactions with a stationary        phase. The stationary phase can be made of chiral material or        the mobile phase can contain an additional chiral material to        provoke the differing interactions;    -   xi) chiral gas chromatography—a technique whereby the racemate        is volatilized and enantiomers are separated by virtue of their        differing interactions in the gaseous mobile phase with a column        containing a fixed non-racemic chiral adsorbent phase;    -   xii) extraction with chiral solvents—a technique whereby the        enantiomers are separated by virtue of preferential dissolution        of one enantiomer into a particular chiral solvent;    -   xiii) transport across chiral membranes—a technique whereby a        racemate is placed in contact with a thin membrane barrier. The        barrier typically separates two miscible fluids, one containing        the racemate, and a driving force such as concentration or        pressure differential causes preferential transport across the        membrane barrier. Separation occurs as a result of the        non-racemic chiral nature of the membrane which allows only one        enantiomer of the racemate to pass through.        II. Definitions

The term “alkyl”, as used herein, unless otherwise specified, refers toa saturated straight, branched, or cyclic, primary, secondary, ortertiary hydrocarbon of typically C₁ to C₁₀, and specifically includesmethyl, CF₃, CCl₃, CFCl₂, CF₂Cl, ethyl, CH₂CF₃, CF₂CF₃, propyl,isopropyl, cyclopropyl, butyl, isobutyl, secbutyl, t-butyl, pentyl,cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl,cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl. The term includes both substituted and unsubstitutedalkyl groups, and particularly includes halogenated alkyl groups, andeven more particularly fluorinated alkyl groups. Non-limiting examplesof moieties with which the alkyl group can be substituted are selectedfrom the group consisting of halogen (fluoro, chloro, bromo or iodo),hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano,sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate,either unprotected, or protected as necessary, as known to those skilledin the art, for example, as taught in Greene, et al., Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991, herebyincorporated by reference.

The term “lower alkyl”, as used herein, and unless otherwise specified,refers to a C₁ to C₄ saturated straight, branched, or if appropriate, acyclic (for example, cyclopropyl) alkyl group, including bothsubstituted and unsubstituted moieties.

The term “alkylamino” or “arylamino” refers to an amino group that hasone or two alkyl or aryl substituents, respectively. Unless otherwisespecifically stated in this application, when alkyl is a suitablemoiety, lower alkyl is preferred. Similarly, when alkyl or lower alkylis a suitable moiety, unsubstituted alkyl or lower alkyl is preferred.

The term “protected” as used herein and unless otherwise defined refersto a group that is added to an oxygen, nitrogen, or phosphorus atom toprevent its further reaction or for other purposes. A wide variety ofoxygen and nitrogen protecting groups are known to those skilled in theart of organic synthesis.

The term “aryl”, as used herein, and unless otherwise specified, refersto phenyl, biphenyl, or naphthyl, and preferably phenyl. The termincludes both substituted and unsubstituted moieties. The aryl group canbe substituted with any described moiety, including, but not limited to,one or more moieties selected from the group consisting of halogen(fluoro, chloro, bromo or iodo), hydroxyl, amino, alkylamino, arylamino,alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,phosphate, or phosphonate, either unprotected, or protected asnecessary, as known to those skilled in the art, for example, as taughtin Greene, et al., Protective Groups in Organic Synthesis, John Wileyand Sons, Second Edition, 1991.

The term “alkaryl” or “alkylaryl” refers to an alkyl group with an arylsubstituent. The term aralkyl or arylalkyl refers to an aryl group withan alkyl substituent.

The term “halo”, as used herein, includes chloro, bromo, iodo, andfluoro.

The term “purine” or “pyrimidine” base includes, but is not limited to,adenine, N⁶-alkylpurines, N⁶-acylpurines (wherein acyl is C(O)(alkyl,aryl, alkylaryl, or arylalkyl), N⁶-benzylpurine, N⁶-halopurine,N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine, N⁶-hydroxyalkylpurine, N⁶-alkylaminopurine, N⁶-thioalkyl purine, N²-alkylpurines,N²-alkyl-6-thiopurines, thymine, cytosine, 5-fluorocytosine,5-methylcytosine, 6-azapyrimidine, including 6-azacytosine, 2- and/or4-mercaptopyrmidine, uracil, 5-halouracil, including 5-fluorouracil,C⁵-alkylpyrimidines, C⁵-benzylpyrimidines, C⁵-halopyrimidines,C⁵-vinylpyrimidine, C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine,C⁵-hydroxyalkyl purine, C⁵-amidopyrimidine, C⁵-cyanopyrimidine,C⁵-iodopyrimidine, C⁶-iodo-pyrimidine, C⁵-Br-vinyl pyrimidine,C⁶-Br-vinyl pyrimidine, C⁵-nitropyrimidine, C⁵-amino-pyrimidine,N²-alkylpurines, N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, andpyrazolopyrimidinyl. Purine bases include, but are not limited to,guanine, adenine, hypoxanthine, 2,6-diaminopurine, and 6-chloropurine.Functional oxygen and nitrogen groups on the base can be protected asnecessary or desired. Suitable protecting groups are well known to thoseskilled in the art, and include trimethylsilyl, dimethylhexylsilyl,t-butyldimethylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups,and acyl groups such as acetyl and propionyl, methanesulfonyl, andp-toluenesulfonyl.

The term “acyl” or “O-linked ester” refers to a group of the formulaC(O)R′, wherein R′ is an straight, branched, or cyclic alkyl (includinglower alkyl), carboxylate reside of amino acid, aryl including phenyl,alkaryl, aralkyl including benzyl, alkoxyalkyl including methoxymethyl,aryloxyalkyl such as phenoxymethyl; or substituted alkyl (includinglower alkyl), aryl including phenyl optionally substituted with chloro,bromo, fluoro, iodo, C₁ to C₄ alkyl or C₁ to C₄ alkoxy, sulfonate esterssuch as alkyl or aralkyl sulphonyl including methanesulfonyl, the mono,di or triphosphate ester, trityl or monomethoxy-trityl, substitutedbenzyl, alkaryl, aralkyl including benzyl, alkoxyalkyl includingmethoxymethyl, aryloxyalkyl such as phenoxymethyl. Aryl groups in theesters optimally comprise a phenyl group. In particular, acyl groupsinclude acetyl, trifluoroacetyl, methylacetyl, cyclpropylacetyl,propionyl, butyryl, hexanoyl, heptanoyl, octanoyl, neo-heptanoyl,phenylacetyl, 2-acetoxy-2-phenylacetyl, diphenylacetyl,α-methoxy-α-trifluoromethyl-phenylacetyl, bromoacetyl,2-nitro-benzeneacetyl, 4-chloro-benzeneacetyl,2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl, trimethylacetyl,chlorodifluoroacetyl, perfluoroacetyl, fluoroacetyl,bromodifluoroacetyl, methoxyacetyl, 2-thiopheneacetyl,chlorosulfonylacetyl, 3-methoxyphenylacetyl, phenoxyacetyl,tert-butylacetyl, trichloroacetyl, monochloro-acetyl, dichloroacetyl,7H-dodecafluoro-heptanoyl, perfluoro-heptanoyl,7H-dodeca-fluoroheptanoyl, 7-chlorododecafluoro-heptanoyl,7-chloro-dodecafluoro-heptanoyl, 7H-dodecafluoroheptanoyl,7H-dodeca-fluoroheptanoyl, nona-fluoro-3,6-dioxa-heptanoyl,nonafluoro-3,6-dioxaheptanoyl, perfluoroheptanoyl, methoxybenzoyl,methyl 3-amino-5-phenylthiophene-2-carboxyl,3,6-dichloro-2-methoxy-benzoyl, 4-(1,1,2,2-tetrafluoro-ethoxy)-benzoyl,2-bromo-propionyl, omega-aminocapryl, decanoyl, n-pentadecanoyl,stearyl, 3-cyclopentyl-propionyl, 1-benzene-carboxyl, O-acetylmandelyl,pivaloyl acetyl, 1-adamantane-carboxyl, cyclohexane-carboxyl,2,6-pyridinedicarboxyl, cyclopropane-carboxyl, cyclobutane-carboxyl,perfluorocyclohexyl carboxyl, 4-methylbenzoyl, chloromethyl isoxazolylcarbonyl, perfluorocyclohexyl carboxyl, crotonyl,1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,1-pyrrolidinecarbonyl, 4-phenylbenzoyl.

The term “amino acid” includes naturally occurring and synthetic α, β γor δ amino acids, and includes but is not limited to, amino acids foundin proteins, i.e. glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan, proline, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,arginine and histidine. In a preferred embodiment, the amino acid is inthe L-configuration. Alternatively, the amino acid can be a derivativeof alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl, phenylalaninyl,tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl,tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl,argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl,β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl,β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl,β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl orβ-histidinyl.

As used herein, the term “substantially free of” or “substantially inthe absence of” refers to a nucleoside composition that includes atleast 85 or 90% by weight, preferably 95%, 98%, 99% or 100% by weight,of the designated enantiomer of that nucleoside. In a preferredembodiment, in the methods and compounds of this invention, thecompounds are substantially free of enantiomers.

Similarly, the term “isolated” refers to a nucleoside composition thatincludes at least 85, 90%, 95%, 98%, 99% to 100% by weight, of thenucleoside, the remainder comprising other chemical species orenantiomers.

The term “host”, as used herein, refers to an unicellular ormulticellular organism in which the virus can replicate, including celllines and animals, and preferably a human. Alternatively, the host canbe carrying a part of the Flaviviridae viral genome, whose replicationor function can be altered by the compounds of the present invention.The term host specifically refers to infected cells, cells transfectedwith all or part of the Flaviviridae genome and animals, in particular,primates (including chimpanzees) and humans. In most animal applicationsof the present invention, the host is a human patient. Veterinaryapplications, in certain indications, however, are clearly anticipatedby the present invention (such as chimpanzees).

The term “pharmaceutically acceptable salt or prodrug” is usedthroughout the specification to describe any pharmaceutically acceptableform (such as an ester, phosphate ester, salt of an ester or a relatedgroup) of a nucleoside compound which, upon administration to a patient,provides the nucleoside compound. Pharmaceutically acceptable saltsinclude those derived from pharmaceutically acceptable inorganic ororganic bases and acids. Suitable salts include those derived fromalkali metals such as potassium and sodium, alkaline earth metals suchas calcium and magnesium, among numerous other acids well known in thepharmaceutical art. Pharmaceutically acceptable prodrugs refer to acompound that is metabolized, for example hydrolyzed or oxidized, in thehost to form the compound of the present invention. Typical examples ofprodrugs include compounds that have biologically labile protectinggroups on a functional moiety of the active compound. Prodrugs includecompounds that can be oxidized, reduced, aminated, deaminated,hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated,dealkylated, acylated, deacylated, phosphorylated, dephosphorylated toproduce the active compound. The compounds of this invention possessantiviral activity against a Flaviviridae, or are metabolized to acompound that exhibits such activity.

III. Prodrugs and Derivatives

Pharmaceutically Aceptable Salts

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compound as apharmaceutically acceptable salt may be appropriate. Examples ofpharmaceutically acceptable salts are organic acid addition salts formedby addition of acids, which form a physiological acceptable anion, forexample, tosylate, methanesulfonate, acetate, citrate, malonate,tartarate, succinate, benzoate, ascorate, α-ketoglutarate,α-glycerophosphate, formate, fumarate, propionate, glycolate, lactate,pyruvate, oxalate, maleate, and salicylate. Suitable inorganic salts mayalso be formed, including, sulfate, nitrate, bicarbonate, carbonatesalts, hydrobromate and phosphoric acid. In a preferred embodiment, thesalt is a mono- or di-hydrochloride salt.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made. In one embodiment, the saltis a hydrochloride salt of the compound. In a further embodiment, thepharmaceutically acceptable salt is a dihydrochloride salt.

Nucleotide Prodrug Formulations

The nucleosides described herein can be administered as a nucleotideprodrug to increase the activity, bioavailability, stability orotherwise alter the properties of the nucleoside. A number of nucleotideprodrug ligands are known. In general, alkylation, acylation or otherlipophilic modification of the mono-, di- or triphosphate of thenucleoside reduces polarity and allows passage into cells. Examples ofsubstituent groups that can replace one or more hydrogens on thephosphate moiety are alkyl, aryl, steroids, carbohydrates, includingsugars, 1,2-diacylglycerol and alcohols.

In an alternative embodiment, the compound is administered as aphosphonate, phosphorothioate or SATE derivative.

Many are described in R. Jones and N. Bischoferger, Antiviral Research,1995, 27:1-17. Any of these can be used in combination with thedisclosed nucleosides to achieve a desired effect. Nonlimiting examplesof U.S. patents that disclose suitable lipophilic substituents that canbe covalently incorporated into the nucleoside, preferably at the 5′-OHposition of the nucleoside or lipophilic preparations, include U.S. Pat.No. 5,149,794 (Sep. 22, 1992, Yatvin et al.); U.S. Pat. No. 5,194,654(Mar. 16, 1993, Hostetler et al., U.S. Pat. No. 5,223,263 (Jun. 29,1993, Hostetler et al.); U.S. Pat. No. 5,256,641 (Oct. 26, 1993, Yatvinet al.); U.S. Pat. No. 5,411,947 (May 2, 1995, Hostetler et al.); U.S.Pat. No. 5,463,092 (Oct. 31, 1995, Hostetler et al.); U.S. Pat. No.5,543,389 (Aug. 6, 1996, Yatvin et al.); U.S. Pat. No. 5,543,390 (Aug.6, 1996, Yatvin et al.); U.S. Pat. No. 5,543,391 (Aug. 6, 1996, Yatvinet al.); and U.S. Pat. No. 5,554,728 (Sep. 10, 1996; Basava et al.), allof which are incorporated herein by reference. Foreign patentapplications that disclose lipophilic substituents that can be attachedto the nucleosides of the present invention, or lipophilic preparations,include WO 89/02733, WO 90/00555, WO 91/16920, WO 91/18914, WO 93/00910,WO 94/26273, WO 96/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721.

The active nucleoside can also be provided as a 2′, 3′ and/or5′-phosphoether lipid or a 2′, 3′ and/or 5′-ether lipid, as disclosed inthe following references, which are incorporated by reference herein:Kucera, L. S., N. Iyer, et al. 1990 AIDS Res. Hum. Retro Viruses.6:491-501; Piantadosi, C., J. Marasco C. 1991 J. Med. Chem.34:1408.1414; Hosteller, K. Y., D. D. Richman, et al. 1992 Antimicrob.Agents Chemother. 36:2025.2029; Hosetler, K. Y., L. M. Stuhmiller, 1990.J. Biol. Chem. 265:61127.

Nonlimiting examples of U.S. patents that disclose suitable lipophilicsubstituents that can be covalently incorporated into the nucleoside,preferably at the 2′, 3′ and/or 5′-OH position of the nucleoside orlipophilic preparations, include U.S. Pat. No. 5,149,794 (Sep. 22, 1992,Yatvin et al.); U.S. Pat. No. 5,194,654 (Mar. 16, 1993, Hostetler etal., U.S. Pat. No. 5,223,263 (Jun. 29, 1993, Hostetler et al.); U.S.Pat. No. 5,256,641 (Oct. 26, 1993, Yatvin et al.); U.S. Pat. No.5,411,947 (May 2, 1995, Hostetler et al.); U.S. Pat. No. 5,463,092 (Oct.31, 1995, Hostetler et al.); U.S. Pat. No. 5,543,389 (Aug. 6, 1996,Yatvin et al.); U.S. Pat. No. 5,543,390 (Aug. 6, 1996, Yatvin et al.);U.S. Pat. No. 5,543,391 (Aug. 6, 1996, Yatvin et al.); and U.S. Pat. No.5,554,728 (Sep. 10, 1996; Basava et al.), all of which are incorporatedherein by reference. Foreign patent applications that discloselipophilic substituents that can be attached to the nucleosides of thepresent invention, or lipophilic preparations, include WO 89/02733, WO90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO 94/26273, WO96/15132, EP 0 350 287, EP 93917054.4, and WO 91/19721.

Aryl esters, especially phenyl esters, are also provided. Nonlimitingexamples are disclosed in DeLambert et al., J. Med. Chem. 37: 498(1994). Phenyl esters containing a carboxylic ester ortho to thephosphate are also provided. Khamnei and Torrence, J. Med. Chem.;39:4109-4115 (1996). In particular, benzyl esters, which generate theparent compound, in some cases using substituents at the ortho- orpara-position to accelerate hydrolysis, are provided. Examples of thisclass of prodrugs are described by Mitchell et al., J. Chem. Soc. PerkinTrans. 12345 (1992); Brook, et al. WO 91/19721; and Glazier et al. WO91/19721.

Cyclic phosphonate esters are also provided. Nonlimiting examples aredisclosed in Hunston et al., J. Med. Chem. 27: 440-444 (1984) andStarrett et al. J. Med. Chem. 37: 1857-1864 (1994). Additionally, cyclic3′,5′-phosphate esters are provided. Nonlimiting examples are disclosedin Meier et al. J. Med. Chem. 22: 811-815 (1979). Cyclic 1′,3′-propanylphosphonate and phosphate esters, such as ones containing a fused arylring, i.e. the cyclosaligenyl ester, are also provided (Meier et al.,Bioorg. Med. Chem. Lett. 7: 99-104 (1997)). Unsubstituted cyclic1′,3′-propanyl esters of the monophosphates are also provided (Farquharet al., J. Med. Chem. 26: 1153 (1983); Farquhar et al., J. Med. Chem.28: 1358 (1985)) were prepared. In addition, cyclic 1′,3′-propanylesters substituted with a pivaloyloxy methyloxy group at C-1′ areprovided (Freed et al., Biochem. Pharmac. 38: 3193 (1989); Biller etal., U.S. Pat. No. 5,157,027).

Cyclic phosphoramidates are known to cleave in vivo by an oxidativemechanism. Therefore, in one embodiment of the present invention, avariety of substituted 1′,3′ propanyl cyclic phosphoramidates areprovided. Non-limiting examples are disclosed by Zon, Progress in Med.Chem. 19, 1205 (1982). Additionally, a number of 2′- and 3′-substitutedproesters are provided. 2′-Substituents include methyl, dimethyl, bromo,trifluoromethyl, chloro, hydroxy, and methoxy; 3′-substituents includingphenyl, methyl, trifluoromethyl, ethyl, propyl, i-propyl, andcyclohexyl. A variety of 1′-substituted analogs are also provided.

Cyclic esters of phosphorus-containing compounds are also provided.Non-limiting examples are described in the following:

-   -   [1] di and tri esters of phosphoric acids as reported in        Nifantyev et al., Phosphorus, Sulfur Silicon and Related        Eelements, 113: 1 (1996); Wijnberg et al., EP-180276 A1;    -   [2] phosphorus (III) acid esters. Kryuchkov et al., Izv. Akad.        Nauk SSSR, Ser. Khim. 6: 1244 (1987). Some of the compounds were        claimed to be useful for the asymmetric synthesis of L-Dopa        precursors. Sylvain et al., DE3512781 A1;    -   [3] phosphoramidates. Shih et al., Bull. Inst. Chem. Acad. Sin,        41: 9 (1994); Edmundson et al., J. Chem. Res. Synop. 5: 122        (1989); and    -   [4] phosphonates. Neidlein et al., Heterocycles 35: 1185 (1993).

Further, nonlimiting examples of U.S. and International PatentApplications that disclose suitable cyclic phosphoramidate prodrugsinclude U.S. Pat. No. 6,312,662; WO 99/45016; WO 00/52015; WO 01/47935;and WO 01/18013 to Erion, et al. from Metabasis Therapeutics, Inc.Specifically, prodrugs of Formula A below are provided:

wherein:

-   -   together V and Z are connected via an additional 3-5 atoms to        form a cyclic group containing 5-7 atoms, optionally 1        heteroatom, substituted with hydroxy, acyloxy,        alkoxycarbonyloxy, or aryloxycarbonyloxy attached to a carbon        atom that is three atoms from both O groups attached to the        phosphorus; or    -   together V and Z are connected via an additional 3-5 atoms to        form a cyclic group, optionally containing 1 heteroatom, that is        fused to an aryl group at the beta and gamma position to the O        attached to the phosphorus;    -   together V and W are connected via an additional 3 carbon atoms        to form an optionally substituted cyclic group containing 6        carbon atoms and substituted with one substituent selected from        the group consisting of hydroxy, acyloxy, alkoxycarbonyloxy,        alkylthiocarbonyloxy, and aryloxycarbonyloxy, attached to one of        said carbon atoms that is three atoms from an O attached to the        phosphorus;    -   together Z and W are connected via an additional 3-5 atoms to        form a cyclic group, optionally containing one heteroatom, and V        must be aryl, substituted aryl, heteroaryl, or substituted        heteroaryl;    -   together W and W′ are connected via an additional 2-5 atoms to        form a cyclic group, optionally containing 0-2 heteroatoms, and        V must be aryl, substituted aryl, heteroaryl, or substituted        heteroaryl;    -   Z is selected from the group consisting of —CHR²OH,        —CHR²OC(O)R³, —CHR²OC(S)R³, —CHR²OC(S)OR³, —CHR²OC(O)SR³,        —CHR²OCO₂R³, —OR², —SR², —CHR²N₃, —CH²aryl, —CH(aryl)OH,        —CH(CH═CR² ₂)OH, —CH(C.ident.CR²)OH, —R², —NR²², —OCOR³,        —OCO₂R³, —SCOR³, —SCO₂R³, —NHCOR², —NHCO₂R³, —CH₂ NHaryl,        —(CH₂), —OR¹², and —(CH₂)_(p)—SR¹²;    -   p is an integer 2 or 3;    -   with the provisos that:        -   a) V, Z, W, W′ are not all —H; and        -   b) when Z is —R², then at least one of V, W, and W′ is not            —H, alkyl, aralkyl, or alicyclic;    -   R² is selected from the group consisting of R³ and —H;    -   R³ is selected from the group consisting of alkyl, aryl,        alicyclic, and aralkyl;    -   R¹² is selected from the group consisting of —H, and lower acyl;    -   M is the biologically active agent, and that is attached to the        phosphorus in Formula A via the 2′, 3′ and/or 5′-hydroxyl.        IV. Combination or Alternation Therapy

The active compounds of the present invention can be administered incombination or alternation with another anti-flavivirus or pestivirusagent, or in particular an anti-HCV agent. In combination therapy,effective dosages of two or more agents are administered together,whereas in alternation or sequential-step therapy, an effective dosageof each agent is administered serially or sequentially. The dosagesgiven will depend on absorption, inactivation and excretion rates of thedrug as well as other factors known to those of skill in the art. It isto be noted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens and schedules should beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions. In preferred embodiments, an anti-HCV (oranti-pestivirus or anti-flavivirus) compound that exhibits an EC₅₀ of10-15 μM, or preferably less than 1-5 μM, is desirable.

It has been recognized that drug-resistant variants of flaviviruses,pestiviruses or HCV can emerge after prolonged treatment with anantiviral agent. Drug resistance most typically occurs by mutation of agene that encodes for an enzyme used in viral replication. The efficacyof a drug against the viral infection can be prolonged, augmented, orrestored by administering the compound in combination or alternationwith a second, and perhaps third, antiviral compound that induces adifferent mutation from that caused by the principle drug.Alternatively, the pharmacokinetics, biodistribution or other parameterof the drug can be altered by such combination or alternation therapy.In general, combination therapy is typically preferred over alternationtherapy because it induces multiple simultaneous stresses on the virus.

Any of the viral treatments described in the Background of the Inventioncan be used in combination or alternation with the compounds describedin this specification. Nonlimiting examples include:

1) Protease Inhibitors

Examples include substrate-based NS3 protease inhibitors (Attwood etal., Antiviral peptide derivatives, PCT WO 98/22496, 1998; Attwood etal., Antiviral Chemistry and Chemotherapy 1999, 10, 259-273; Attwood etal., Preparation and use of amino acid derivatives as anti-viral agents,German Patent Pub. DE 19914474; Tung et al. Inhibitors of serineproteases, particularly hepatitis C virus NS3 protease, PCT WO98/17679), including alphaketoamides and hydrazinoureas, and inhibitorsthat terminate in an electrophile such as a boronic acid or phosphonate(Llinas-Brunet et al, Hepatitis C inhibitor peptide analogues, PCT WO99/07734); Non-substrate-based NS3 protease inhibitors such as2,4,6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al.,Biochemical and Biophysical Research Communications, 1997, 238, 643-647;Sudo K. et al. Antiviral Chemistry and Chemotherapy, 1998, 9, 186),including RD3-4082 and RD3-4078, the former substituted on the amidewith a 14 carbon chain and the latter processing a para-phenoxyphenylgroup; and Sch 68631, a phenanthrenequinone, an HCV protease inhibitor(Chu M. et al., Tetrahedron Letters 37:7229-7232, 1996).

Sch 351633, isolated from the fungus Penicillium griseofulvum, wasidentified as a protease inhibitor (Chu M. et al., Bioorganic andMedicinal Chemistry Letters 9:1949-1952). Eglin c, isolated from leech,is a potent inhibitor of several serine proteases such as S. griseusproteases A and B, α-chymotrypsin, chymase and subtilisin. Qasim M. A.et al., Biochemistry 36:1598-1607, 1997.

U.S. patents disclosing protease inhibitors for the treatment of HCVinclude, for example, U.S. Pat. No. 6,004,933 to Spruce et al. whichdiscloses a class of cysteine protease inhibitors for inhibiting HCVendopeptidase 2; U.S. Pat. No. 5,990,276 to Zhang et al. which disclosessynthetic inhibitors of hepatitis C virus NS3 protease; U.S. Pat. No.5,538,865 to Reyes et a; WO 02/008251 to Corvas International, Inc, andWO 02/08187 and WO 02/008256 to Schering Corporation. HCV inhibitortripeptides are disclosed in U.S. Pat. Nos. 6,534,523, 6,410,531, and6,420,380 to Boehringer Ingelheim and WO 02/060926 to Bristol MyersSquibb. Diaryl peptides as NS3 serine protease inhibitors of HCV aredisclosed in WO 02/48172 to Schering Corporation. Imidazoleidinones asNS3 serine protease inhibitors of HCV are disclosed in WO 02/08198 toSchering Corporation and WO 02/48157 to Bristol Myers Squibb. WO98/17679 to Vertex Pharmaceuticals and WO 02/48116 to Bristol MyersSquibb also disclose HCV protease inhibitors.

-   -   2) Thiazolidine derivatives which show relevant inhibition in a        reverse-phase HPLC assay with an NS3/4A fusion protein and        NS5A/5B substrate (Sudo K. et al., Antiviral Research, 1996, 32,        9-18), especially compound RD-1-6250, possessing a fused        cinnamoyl moiety substituted with a long alkyl chain, RD4 6205        and RD4 6193;    -   3) Thiazolidines and benzanilides identified in Kakiuchi N. et        al. J. EBS Letters 421, 217-220; Takeshita N. et al. Analytical        Biochemistry, 1997, 247, 242-246;    -   4) A phenan-threnequinone possessing activity against protease        in a SDS-PAGE and autoradiography assay isolated from the        fermentation culture broth of Streptomyces sp., Sch 68631        (Chu M. et al., Tetrahedron Letters, 1996, 37, 7229-7232), and        Sch 351633, isolated from the fungus Penicillium griseofulvum,        which demonstrates activity in a scintillation proximity assay        (Chu M. et al., Bioorganic and Medicinal Chemistry Letters 9,        1949-1952);    -   5) Helicase inhibitors (Diana G. D. et al., Compounds,        compositions and methods for treatment of hepatitis C, U.S. Pat.        No. 5,633,358; Diana G. D. et al., Piperidine derivatives,        pharmaceutical compositions thereof and their use in the        treatment of hepatitis C, PCT WO 97/36554);    -   6) Nucleotide polymerase inhibitors and gliotoxin (Ferrari R. et        al. Journal of Virology, 1999, 73, 1649-1654), and the natural        product cerulenin (Lohmann V. et al., Virology, 1998, 249,        108-118);    -   7) Antisense phosphorothioate oligodeoxynucleotides (S-ODN)        complementary to sequence stretches in the 5′ non-coding region        (NCR) of the virus (Alt M. et al., Hepatology, 1995, 22,        707-717), or nucleotides 326-348 comprising the 3′ end of the        NCR and nucleotides 371-388 located in the core coding region of        the HCV RNA (Alt M. et al., Archives of Virology, 1997, 142,        589-599; Galderisi U. et al., Journal of Cellular Physiology,        1999, 181, 251-257);    -   8) Inhibitors of IRES-dependent translation (Ikeda N et al.,        Agent for the prevention and treatment of hepatitis C, Japanese        Patent Pub. JP-08268890; Kai Y. et al. Prevention and treatment        of viral diseases, Japanese Patent Pub. JP-10101591);    -   9) Ribozymes, such as nuclease-resistant ribozymes        (Maccjak, D. J. et al., Hepatology 1999, 30, abstract 995) and        those disclosed in U.S. Pat. No. 6,043,077 to Barber et al., and        U.S. Pat. Nos. 5,869,253 and 5,610,054 to Draper et al.; and    -   10) Nucleoside analogs have also been developed for the        treatment of Flaviviridae infections.    -   11) Any of the compounds described by Idenix Pharmaceuticals in        International Publication Nos. WO 01/90121 and WO 01/92282;    -   12) Other patent applications disclosing the use of certain        nucleoside analogs to treat hepatitis C virus include:        PCT/CA00/01316 (WO 01/32153; filed Nov. 3, 2000) and        PCT/CA01/00197 (WO 01/60315; filed Feb. 19, 2001) filed by        BioChem Pharma, Inc. (now Shire Biochem, Inc.); PCT/US02/01531        (WO 02/057425; filed Jan. 18, 2002) and PCT/US02/03086 (WO        02/057287; filed Jan. 18, 2002) filed by Merck & Co., Inc.,        PCT/EP01/09633 (WO 02/18404; published Aug. 21, 2001) filed by        Roche, and PCT Publication Nos. WO 01/79246 (filed Apr. 13,        2001), WO 02/32920 (filed Oct. 18, 2001) and WO 02/48165 by        Pharmasset, Ltd.    -   13) PCT Publication No. WO 99/43691 to Emory University,        entitled “2′-Fluoronucleosides” discloses the use of certain        2′-fluoronucleosides to treat HCV.

14) Other miscellaneous compounds including 1-amino-alkylcyclohexanes(U.S. Pat. No. 6,034,134 to Gold et al.), alkyl lipids (U.S. Pat. No.5,922,757 to Chojkier et al.), vitamin E and other antioxidants (U.S.Pat. No. 5,922,757 to Chojkier et al.), squalene, amantadine, bile acids(U.S. Pat. No. 5,846,964 to Ozeki et al.),N-(phosphonoacetyl)-L-aspartic acid, (U.S. Pat. No. 5,830,905 to Dianaet al.), benzenedicarboxamides (U.S. Pat. No. 5,633,388 to Diana etal.), polyadenylic acid derivatives (U.S. Pat. No. 5,496,546 to Wang etal.), 2′,3′-dideoxyinosine (U.S. Pat. No. 5,026,687 to Yarchoan et al.),benzimidazoles (U.S. Pat. No. 5,891,874 to Colacino et al.), plantextracts (U.S. Pat. No. 5,837,257 to Tsai et al., U.S. Pat. No.5,725,859 to Omer et al., and U.S. Pat. No. 6,056,961), and piperidenes(U.S. Pat. No. 5,830,905 to Diana et al.).

-   -   15) Any other compounds currently in preclinical or clinical        development for treatment of hepatitis C virus including:        Interleukin-10 by Schering-Plough, IP-501 by Interneuron,        Merimebodib (VX-497) by Vertex, AMANTADINE® (Symmetrel) by Endo        Labs Solvay, HEPTAZYME® by RPI, IDN-6556 by Idun Pharma.,        XTL-002 by XTL., HCV/MF59 by Chiron, CIVACIR® (Hepatitis C        Immune Globulin) by NABI, LEVOVIRIN® by ICN/Ribapharm,        VIRAMIDINE® by ICN/Ribapharm, ZADAXINS (thymosin alpha-1) by Sci        Clone, thymosin plus pegylated interferon by Sci Clone, CEPLENE®        (histamine dihydrochloride) by Maxim, VX 950/LY 570310 by        Vertex/Eli Lilly, ISIS 14803 by Isis Pharmaceutical/Elan,        IDN-6556 by Idun Pharmaceuticals, Inc., JTK 003 by AKROS Pharma,        BILN-2061 by Boehringer Ingelheim, CellCept (mycophenolate        mofetil) by Roche, T67, a β-tubulin inhibitor, by Tularik, a        therapeutic vaccine directed to E2 by Innogenetics, FK788 by        Fujisawa Healthcare, Inc., 1 dB 1016 (Siliphos, oral        silybin-phosphatdylcholine phytosome), RNA replication        inhibitors (VP50406) by ViroPharma/Wyeth, therapeutic vaccine by        Intercell, therapeutic vaccine by Epimmune/Genencor, IRES        inhibitor by Anadys, ANA 245 and ANA 246 by Anadys,        immunotherapy (Therapore) by Avant, protease inhibitor by        Corvas/SChering, helicase inhibitor by Vertex, fusion inhibitor        by Trimeris, T cell therapy by CellExSys, polymerase inhibitor        by Biocryst, targeted RNA chemistry by PTC Therapeutics,        Dication by Immtech, Int., protease inhibitor by Agouron,        protease inhibitor by Chiron/Medivir, antisense therapy by AVI        BioPharma, antisense therapy by Hybridon, hemopurifier by        Aethlon Medical, therapeutic vaccine by Merix, protease        inhibitor by Bristol-Myers Squibb/Axys, Chron-VacC, a        therapeutic vaccine, by Tripep, UT 231B by United Therapeutics,        protease, helicase and polymerase inhibitors by Genelabs        Technologies, IRES inhibitors by Immusol, R803 by Rigel        Pharmaceuticals, INFERGEN® (interferon alphacon-1) by InterMune,        OMNIFERON® (natural interferon) by Viragen, ALBUFERON® by Human        Genome Sciences, REBIF® (interferon beta-1a) by Ares-Serono,        Omega Interferon by BioMedicine, Oral Interferon Alpha by        Amarillo Biosciences, interferon gamma, interferon tau, and        Interferon gamma-1b by InterMune.        V. Pharmaceutical Compositions

Hosts, including humans, infected with pestivirus, flavivirus, HCV oranother organism replicating through a RNA-dependent RNA viralpolymerase, or for treating any other disorder described herein, can betreated by administering to the patient an effective amount of theactive compound or a pharmaceutically acceptable prodrug or salt thereofin the presence of a pharmaceutically acceptable carrier or dilutent.The active materials can be administered by any appropriate route, forexample, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid or solid form.

A preferred dose of the compound for pestivirus, flavivirus or HCVinfection or any other condition described herein will be in the rangefrom about 1 to 50 mg/kg, preferably 1 to 20 mg/kg, of body weight perday, more generally 0.1 to about 100 mg per kilogram body weight of therecipient per day. Lower doses may be preferable, for example doses of0.5-100 mg, 0.5-50 mg, 0.5-10 mg, or 0.5-5 mg per kilogram body weightper day. Even lower doses may be useful, and thus ranges can alsoinclude from 0.1-0.5 mg per kilogram body weight per day. The effectivedosage range of the pharmaceutically acceptable salts and prodrugs canbe calculated based on the weight of the parent nucleoside to bedelivered. If the salt or prodrug exhibits activity in itself, theeffective dosage can be estimated as above using the weight of the saltor prodrug, or by other means known to those skilled in the art.

The compound is conveniently administered in a unit of any suitabledosage form, including but not limited to one containing 7 to 3000 mg,preferably 70 to 1400 mg of active ingredient per unit dosage form. Anoral dosage of 50-1000 mg is usually convenient, including in one ormultiple dosages of 50, 100, 200, 250, 300, 400, 500, 600, 700, 800, 900or 1000 mgs. Lower doses may be preferable, for example from 10-100 or1-50 mg. Also contemplated are doses of 0.1-50 mg, or 0.1-20 mg or0.1-10.0 mg. Furthermore, lower doses may be utilized in the case ofadministration by a non-oral route, as, for example, by injection orinhalation.

Ideally the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.2 to 70 μM,preferably about 1.0 to 10 μM. This may be achieved, for example, by theintravenous injection of a 0.1 to 5% solution of the active ingredient,optionally in saline, or administered as a bolus of the activeingredient.

The concentration of active compound in the drug composition will dependon absorption, inactivation and excretion rates of the drug as well asother factors known to those of skill in the art. It is to be noted thatdosage values will also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedcomposition. The active ingredient may be administered at once, or maybe divided into a number of smaller doses to be administered at varyingintervals of time.

A preferred mode of administration of the active compound is oral. Oralcompositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

The compound can be administered as a component of an elixir,suspension, syrup, wafer, chewing gum or the like. A syrup may contain,in addition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors.

The compound or a pharmaceutically acceptable prodrug or salts thereofcan also be mixed with other active materials that do not impair thedesired action, or with materials that supplement the desired action,such as antibiotics, antifungals, anti-inflammatories, or otherantivirals, including other nucleoside compounds. Solutions orsuspensions used for parenteral, intradermal, sucutaneous, or topicalapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parental preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

In a preferred embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) are also preferred aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811 (which is incorporated herein by reference inits entirety). For example, liposome formulations may be prepared bydissolving appropriate lipid(s) (such as stearoyl phosphatidylethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidylcholine, and cholesterol) in an inorganic solvent that is thenevaporated, leaving behind a thin film of dried lipid on the surface ofthe container. An aqueous solution of the active compound or itsmonophosphate, diphosphate, and/or triphosphate derivatives is thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension.

VI. Processes for the Preparation of Active Compounds

The nucleosides of the present invention can be synthesized by any meansknown in the art. In particular, the synthesis of the presentnucleosides can be achieved by either alkylating the appropriatelymodified sugar, followed by glycosylation or glycosylation followed byalkylation of the nucleoside. The following non-limiting embodimentsillustrate some general methodology to obtain the nucleosides of thepresent invention.

A. General Synthesis of 1′-C-Branched Nucleosides

1′-C-Branched ribonucleosides of the following structure:

wherein BASE is a purine or pyrimidine base as defined herein;R⁷ and R⁹ are independently hydrogen, OR², hydroxy, alkyl (includinglower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), chlorine, bromine, iodine, NO₂, NH₂, —NH(loweralkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;R⁸ and R¹⁰ are independently H, alkyl (including lower alkyl), chlorine,bromine or iodine; alternatively, R⁷ and R⁹, R⁷ and R¹⁰, R⁸ and R⁹, orR⁸ and R¹⁰ can come together to form a pi bond;R¹ and R² are independently H; phosphate (including monophosphate,diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl(including lower acyl); alkyl (including lower alkyl); sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein; alipid, including a phospholipid; an amino acid; a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich is capable of providing a compound wherein R¹ or R² isindependently H or phosphate, for example when administered in vivo;R⁶ is an alkyl, chloro-, bromo-, fluoro-, or iodo-alkyl (i.e. CF₃),alkenyl, or alkynyl (i.e. allyl); andX is O, S, SO₂ or CH₂can be prepared by one of the following general methods.1) Modification from the Lactone

The key starting material for this process is an appropriatelysubstituted lactone. The lactone can be purchased or can be prepared byany known means including standard epimerization, substitution andcyclization techniques. The lactone can be optionally protected with asuitable protecting group, preferably with an acyl or silyl group, bymethods well known to those skilled in the art, as taught by Greene etal. Protective Groups in Organic Synthesis, John Wiley and Sons, SecondEdition, 1991. The protected lactone can then be coupled with a suitablecoupling agent, such as an organometallic carbon nucleophile, such as aGrignard reagent, an organolithium, lithium dialkylcopper or R⁶—SiMe₃ inTBAF with the appropriate non-protic solvent at a suitable temperature,to give the 1′-alkylated sugar.

The optionally activated sugar can then be coupled to the BASE bymethods well known to those skilled in the art, as taught by TownsendChemistry of Nucleosides and Nucleotides, Plenum Press, 1994. Forexample, an acylated sugar can be coupled to a silylated base with aLewis acid, such as tin tetrachloride, titanium tetrachloride ortrimethylsilyltriflate in the appropriate solvent at a suitabletemperature.

Subsequently, the nucleoside can be deprotected by methods well known tothose skilled in the art, as taught by Greene et al. Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991.

In a particular embodiment, the 1′-C-branched ribonucleoside is desired.The synthesis of a ribonucleoside is shown in Scheme 1. Alternatively,deoxyribo-nucleoside is desired. To obtain these nucleosides, the formedribonucleoside can optionally be protected by methods well known tothose skilled in the art, as taught by Greene et al. Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991, andthen the 2′-OH can be reduced with a suitable reducing agent.Optionally, the 2′-hydroxyl can be activated to facilitate reduction;i.e. via the Barton reduction.

2. Alternative Method for the Preparation of 1′-C-Branched Nucleosides

The key starting material for this process is an appropriatelysubstituted hexose. The hexose can be purchased or can be prepared byany known means including standard epimerization, such as alkalinetreatment, substitution and coupling techniques. The hexose can beselectively protected to give the appropriate hexa-furanose, as taughtby Townsend Chemistry of Nucleosides and Nucleotides, Plenum Press,1994.

The 1′-hydroxyl can be optionally activated to a suitable leaving groupsuch as an acyl group or a chloro, bromo, fluoro, iodo via acylation orhalogenation, respectively. The optionally activated sugar can then becoupled to the BASE by methods well known to those skilled in the art,as taught by Townsend Chemistry of Nucleosides and Nucleotides, PlenumPress, 1994. For example, an acylated sugar can be coupled to asilylated base with a Lewis acid, such as tin tetrachloride, titaniumtetrachloride or trimethylsilyltriflate in the appropriate solvent at asuitable temperature. Alternatively, a halo-sugar can be coupled to asilylated base with the presence of trimethylsilyltriflate.

The 1′-CH₂—OH, if protected, can be selectively deprotected by methodswell known in the art. The resultant primary hydroxyl can befunctionalized to yield various C-branched nucleosides. For example, theprimary hydroxyl can be reduced to give the methyl, using a suitablereducing agent. Alternatively, the hydroxyl can be activated prior toreduction to facilitate the reaction; i.e. via the Barton reduction. Inan alternate embodiment, the primary hydroxyl can be oxidized to thealdehyde, then coupled with a carbon nucleophile, such as a Grignardreagent, an organolithium, lithium dialkylcopper or R⁶—SiMe₃ in TBAFwith the appropriate non-protic solvent at a suitable temperature.

In a particular embodiment, the 1′-C-branched ribonucleoside is desired.The synthesis of a ribonucleoside is shown in Scheme 2. Alternatively,deoxyribo-nucleoside is desired. To obtain these nucleosides, the formedribonucleoside can optionally be protected by methods well known tothose skilled in the art, as taught by Greene et al. Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991, andthen the 2′-OH can be reduced with a suitable reducing agent.Optionally, the 2′-hydroxyl can be activated to facilitate reduction;i.e. via the Barton reduction.

In addition, the L-enantiomers corresponding to the compounds of theinvention can be prepared following the same general methods (1 or 2),beginning with the corresponding L-sugar or nucleoside L-enantiomer asstarting material.

B. General Synthesis of 2′-C-Branched Nucleosides

2′-C-Branched ribonucleosides of the following structure:

wherein BASE is a purine or pyrimidine base as defined herein;R⁷ and R⁹ are independently hydrogen, OR², hydroxy, alkyl (includinglower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), chlorine, bromine, iodine, NO₂, NH₂, —NH(loweralkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;R¹⁰ is H, alkyl (including lower alkyl), chlorine, bromine or iodine;alternatively, R⁷ and R⁹, or R⁷ and R¹⁰ can come together to form a pibond;R¹ and R² are independently H; phosphate (including monophosphate,diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl(including lower acyl); alkyl (including lower alkyl); sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein; alipid, including a phospholipid; an amino acid; a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R¹ or R² is independently H or phosphate;R⁶ is an alkyl, chloro-, bromo-, fluoro-, iodo-alkyl (i.e. CF₃),alkenyl, or alkynyl (i.e. allyl); andX is O, S, SO₂ or CH₂can be prepared by one of the following general methods.1. Glycosylation of the Nucleobase with an Appropriately Modified Sugar

The key starting material for this process is an appropriatelysubstituted sugar with a 2′-OH and 2′-H, with the appropriate leavinggroup (LG), for example an acyl group or a chloro, bromo, fluoro oriodo. The sugar can be purchased or can be prepared by any known meansincluding standard epimerization, substitution, oxidation and reductiontechniques. The substituted sugar can then be oxidized with theappropriate oxidizing agent in a compatible solvent at a suitabletemperature to yield the 2′-modified sugar. Possible oxidizing agentsare Jones reagent (a mixture of chromic acid and sulfuric acid),Collins's reagent (dipyridine Cr(VI) oxide, Corey's reagent (pyridiniumchlorochromate), pyridinium dichromate, acid dichromate, potassiumpermanganate, MnO₂, ruthenium tetroxide, phase transfer catalysts suchas chromic acid or permanganate supported on a polymer, Cl₂-pyridine,H₂O₂-ammonium molybdate, NaBrO₂-CAN, NaOCl in HOAc, copper chromite,copper oxide, Raney nickel, palladium acetate, Meerwin-Pondorf-Verleyreagent (aluminum t-butoxide with another ketone) andN-bromosuccinimide.

Then coupling of an organometallic carbon nucleophile, such as aGrignard reagent, an organolithium, lithium dialkylcopper or R⁶—SiMe₃ inTBAF with the ketone with the appropriate non-protic solvent at asuitable temperature, yields the 2′-alkylated sugar. The alkylated sugarcan be optionally protected with a suitable protecting group, preferablywith an acyl or silyl group, by methods well known to those skilled inthe art, as taught by Greene et al. Protective Groups in OrganicSynthesis, John Wiley and Sons, Second Edition, 1991.

The optionally protected sugar can then be coupled to the BASE bymethods well known to those skilled in the art, as taught by TownsendChemistry of Nucleosides and Nucleotides, Plenum Press, 1994. Forexample, an acylated sugar can be coupled to a silylated base with aLewis acid, such as tin tetrachloride, titanium tetrachloride ortrimethylsilyltriflate in the appropriate solvent at a suitabletemperature. Alternatively, a halo-sugar can be coupled to a silylatedbase with the presence of trimethylsilyltriflate.

Subsequently, the nucleoside can be deprotected by methods well known tothose skilled in the art, as taught by Greene et al. Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991.

In a particular embodiment, the 2′-C-branched ribonucleoside is desired.The synthesis of a ribonucleoside is shown in Scheme 3. Alternatively,deoxyribo-nucleoside is desired. To obtain these nucleosides, the formedribonucleoside can optionally be protected by methods well known tothose skilled in the art, as taught by Greene et al. Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991, andthen the 2′-OH can be reduced with a suitable reducing agent.Optionally, the 2′-hydroxyl can be activated to facilitate reduction;i.e. via the Barton reduction.

2. Modification of a Pre-Formed Nucleoside

The key starting material for this process is an appropriatelysubstituted nucleoside with a 2′-OH and 2′-H. The nucleoside can bepurchased or can be prepared by any known means including standardcoupling techniques. The nucleoside can be optionally protected withsuitable protecting groups, preferably with acyl or silyl groups, bymethods well known to those skilled in the art, as taught by Greene etal. Protective Groups in Organic Synthesis, John Wiley and Sons, SecondEdition, 1991.

The appropriately protected nucleoside can then be oxidized with theappropriate oxidizing agent in a compatible solvent at a suitabletemperature to yield the 2′-modified sugar. Possible oxidizing agentsare Jones reagent (a mixture of chromic acid and sulfuric acid),Collins's reagent (dipyridine Cr(VI) oxide, Corey's reagent (pyridiniumchlorochromate), pyridinium dichromate, acid dichromate, potassiumpermanganate, MnO₂, ruthenium tetroxide, phase transfer catalysts suchas chromic acid or permanganate supported on a polymer, Cl₂-pyridine,H₂O₂-ammonium molybdate, NaBrO₂-CAN, NaOCl in HOAc, copper chromite,copper oxide, Raney nickel, palladium acetate, Meerwin-Pondorf-Verleyreagent (aluminum t-butoxide with another ketone) andN-bromosuccinimide.

Subsequently, the nucleoside can be deprotected by methods well known tothose skilled in the art, as taught by GreeneGreene et al. ProtectiveGroups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991.

In a particular embodiment, the 2′-C-branched ribonucleoside is desired.The synthesis of a ribonucleoside is shown in Scheme 4. Alternatively,deoxyribo-nucleoside is desired. To obtain these nucleosides, the formedribonucleoside can optionally be protected by methods well known tothose skilled in the art, as taught by Greene et al._(—) ProtectiveGroups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991,and then the 2′-OH can be reduced with a suitable reducing agent.Optionally, the 2′-hydroxyl can be activated to facilitate reduction;i.e. via the Barton reduction.

3. Synthesis Of β-2′-C-Methyl-Ribofuranosyl Cytidine-3′-O-L-Valine Ester

In one synthesis method, depicted in FIG. 5, the synthesis comprisesreacting cytosine, BSA and SnCl₄/acetonitrile with1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose (FIG. 5, compound 1)to form4-amino-1-(3,4-dibenzoyloxy-5-benzoyloxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one(FIG. 5, compound 2); and reacting (FIG. 5, compound 2 with NaOMe/MeOHto provide4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one(FIG. 5, compound 3), also known as 2-C-methyl-β-D-ribofuranose. The useof cytosine as a starting material rather than benzoyl-cytosine improvesthe “atom economy” of the process and simplifies purification at latersteps.

The next steps in this process comprise reacting (FIG. 5, compound 3)with Me₂NCH(OMe)₂ in DMF to form (FIG. 5, compound 4),N-[1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-2-oxo-1,2-dihydro-pyrimidin-4-yl]-N,N-dimethyl-formamidine,which is the amino-protected form of (FIG. 5, compound 3); reacting(FIG. 5, compound 4) with TBDPSCl and imidazole in DCM to provide the5′-silyl-protected form of (FIG. 5, compound 4) asN′-{1-[5-(tert-butyl-diphenyl-silanyloxymethyl)-3,4-dihydroxy-3-methyl-tetrahydro-furan-2-yl]-2-oxo-1,2-dihydro-pyrimidin-4-yl}-N,N-dimethyl-formamidine(FIG. 5, compound 5), where the use of DCM provides the advantage ofhaving greater control over disilyl by-product formation; reacting (FIG.5, compound 5) with N-Boc-L-valine, EDC and DMAP in DCM at roomtemperature to form 2-tert-butoxycarbonylamino-3-methyl-butyric acid2-(tert-butyl-diphenyl-silanyloxy-methyl)-5-[4-(dimethylamino-methyleneamino)-2-oxo-2H-pyrimidin-1-yl]-4-hydroxy-4-methyl-tetrahydro-furan-3-ylester (FIG. 5, compound 6); removing the silyl and amino-protectinggroups by reacting (FIG. 5, compound 6) with NH₄F in MeOH in thepresence of approximately 10 mole equivalents of ethyl acetate toprevent cleavage of the 3′-O-valinyl ester by liberated ammonia, andrefluxing the mixture to provide2-tert-butoxycarbonylamino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester to provide (FIG. 5, compound 2); and finally, reacting (FIG. 5,compound 2) with HCl in EtOH to provide 2-amino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester, dihydrochloride salt (FIG. 5, compound 8) as a final product.

6. Alternative Synthesis of β-D-2′-C-Methyl-RibofuranosylCytidine-3′-O-L-Valine Ester

In another method to synthesize the compounds of the invention, shown inFIG. 6, benzoylcytosine, BSA and SnCl₄/acetonitrile are reacted with1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose (FIG. 6, compound a)to form4-benzoylamino-1-(3,4-dibenzoyloxy-5-benzoyloxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one(FIG. 6, compound 2a); reacting (FIG. 6, compound A) with NH₃ inmethanol and chromatographically separating the product,4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidin-2-one(FIG. 6, compound 3a), also known as β-D-2′-C-methyl-cytidine; reacting(FIG. 6, compound 2a) with Me₂NCH(OMe)₂ in DMF at room temperature for1.5 hours to formN-[1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-2-oxo-1,2-dihydro-pyrimidin-4-yl]-N,N-dimethyl-formamidine(FIG. 6, compound a); reacting (FIG. 6, compound 4a) with TBDPSCl andpyridine at room temperature for 6 hours to provideN′-{1-[5-(tert-butyl-diphenyl-silanyloxymethyl)-3,4-dihydroxy-3-methyl-tetrahydro-furan-2-yl]-2-oxo-1,2-dihydro-pyrimidin-4-yl}-N,N-dimethyl-formamidine(FIG. 6, compound 1a; reacting (FIG. 6, compound 5a) withN-Boc-L-valine, DEC and DMAP in THF/DMF at room temperature for 2 daysand subjecting the product formed from this reaction to HPLC in order toprovide 2-tert-butoxycarbonylamino-3-methyl-butyric acid2-(tert-butyl-diphenyl-silanyloxy-methyl)-5-[4-(dimethylaminomethyleneamino)-2-oxo-2H-pyrimidin-1-yl]-4-hydroxy-4-methyl-tetrahydro-furan-3-ylester (FIG. 6, compound 6a); refluxing (FIG. 6, compound 6a) with NH₄Fin MeOH for about 3 hours to remove the silyl and amino-protectinggroups, and subjecting the product to chromatographic purification toprovide 2-tert-butoxycarbonylamino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester (FIG. 6, compound 7a); and finally reacting (FIG. 6, compound 7a)with HCl in EtOAc at room temperature to provide2-amino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester, dihydrochloride salt (FIG. 6, compound 8a) as a final product.

The synthesis of 2′-C-methyl-cytidine-3′-O-L-valine ester (val-mcyd) isdepicted in scheme 5 and scheme 6, and described below.

Step 1: Synthesis of Scheme 5, Compound 9:2-C-Methyl-D-ribonic-γ-lactone

De-ionized water (100 mL) was stirred in a 250 mL 3-necked round bottomflask, equipped with an overhead stirrer, a stirring shaft, a digitaltemperature read-out device and an argon line. Argon was bubbled intowater for thirty minutes and D-fructose (20.0 g, 0.111 mole) was addedand the solution became clear in a few minutes. Calcium oxide (12.5 g,0.223 mole) was added in portions over a period of five minutes and themixture was vigorously stirred. An exotherm was observed and reactiontemperature reached 39.6° C. after 10 minutes from the start of thecalcium oxide addition. After about fifteen minutes, the reactionmixture developed a yellow color that deepened with time. After threehours, an aliquot was withdrawn for TLC analysis. The aliquot wasacidified to pH 2 using saturated aqueous solution of oxalic acid. Theresulting white suspension was evaporated under reduced pressure toremove the water. Toluene (2 mL) was added to the residue and themixture was evaporated under reduced pressure (at 45-50° C.) to removeany trace of water. The residual solid was re-constituted in 2 mL of 1:1tetrahydrofuran:methanol mixture. After thorough mixing, the suspensionwas allowed to settle and the supernatant clear solution was spotted forTLC (silica plate was developed in 2% methanol in ethyl acetate andstained in 1% alkaline potassium permanganate dip. The plate was thenheated, using a heat gun, until the appearance of yellowish spots on thepink background). The desired lactone typically appears at an R_(f)value of 0.33 under the above conditions. More polar by-products andunreacted material are detected in the R_(f) value range of 0.0 to 0.2.

Although product formation was observed after 3 hours, the reaction wasallowed to continue for 22 hours during which time the reaction mixturewas stirred at 25° C. At the end of this period, pH of the mixture was13.06. Carbon dioxide gas was bubbled into the reaction mixture forabout 2.5 hours (pH was 7.25). The formed calcium carbonate solid wasremoved by vacuum filtration, filter cake washed with 50 mL ofde-ionized water. The aqueous layers were combined and treated withoxalic acid (5.0 g, 0.056 mole) and the mixture was vigorously stirredat 25° C. for 30 minutes (The initial dark color largely disappeared andthe mixture turned into a milky white slurry). The pH of the mixture atthis stage is typically 2-3. The slurry mixture was stirred at 45-50° C.overnight. The mixture was then evaporated under reduced pressure and at45-50° C. to remove 75 mL of water. Sodium chloride (30 g) andtetrahydrofuran (100 mL) were added to the aqueous slurry (about 75 mL)and the mixture was vigorously stirred at 25° C. for 30 minutes. Thelayers were separated and the aqueous layer was stirred for 10 minuteswith 75 mL of fresh tetrahydrofuran. This process was repeated for threetimes and the tetrahydrofuran solutions were combined and stirred with10 g of anhydrous magnesium sulfate for 30 minutes. The mixture wasfiltered and the magnesium sulfate filter cake was washed with 60 mL oftetrahydrofuran. The filtrate was evaporated under reduced pressure andat 40° C. to give 10.86 g of crude product as a dark orange semisolid.(For scale up runs tetrahydrofuran will be replaced with acetone insteadof evaporation of crude product to dryness). Crude product was stirredwith acetone (20 mL) at 20° C. for 3 hours. Product was collected byvacuum filtration and the filter cake washed with 12 mL of acetone togive the desired product 9 as white crystalline solid. Product was driedin vacuum to give 2.45 g (13.6% yield). Melting point of compound 9:158-162° C. (literature melting point: 160-161° C.). ¹H NMR (DMSO-d₆) δppm 5.69 (s, 1H, exch. With D₂O), 5.41 (d, 1H, exch. With D₂O), 5.00 (t,1H, exch. With D₂O), 4.15 (m, 1H), 3.73 (m, 2H), 3.52 (m, 1H), 1.22 (s,3H). ¹³C NMR (DMSO-d₆) δ ppm 176.44, 82.95, 72.17, 72.02, 59.63, 20.95.(C₆H₁₀O₅: calcd C, 44.45; H, 6.22. Found: C, 44.34; H, 6.30).

Step 2: Synthesis of Scheme 5, Compound 10:2,3,5-Tri-O-benzoyl-2-C-methyl-D-ribonic-γ-lactone

A mixture of lactone 1 (3.0 g, 18.50 mmol.), 4-dimethylaminopyridine(0.45 g, 3.72 mmol.) and triethylamine (25.27 g, 249.72 mmol.) in1,2-dimethoxy ethane(50 mL) was stirred at 25° C. under argon atmospherefor thirty minutes. This white suspension was cooled to 5° C. andbenzoyl chloride (11.7 g, 83.23 mmol.) was added over a period offifteen minutes. The mixture was stirred at 25° C. for two hours. TLCanalysis (silica, 2% methanol in ethyl acetate) indicated completeconsumption of starting material. Ice cold water (100 g) was added tothe reaction mixture and stirring was continued for thirty minutes. Theformed white solids were collected by vacuum filtration and filter cakewashed with cold water (50 mL). This crude product was stirred withtert-butyl methyl ether (60 mL) at 20° C. for thirty minutes, thenfiltered, filter cake washed with tert-butyl methyl ether (25 mL) anddried in vacuum to give 7.33 g (83.4% yield) of compound 10 as a whitesolid in 97.74% purity (HPLC/AUC). Melting point of compound 10:137-140° C. (literature melting point: 141-142° C.). ¹H NMR (CDCl₃) δppm 8.04 (d, 2H), 7.92 (d, 2H), 7.73 (d, 2H), 7.59 (t, 1H), 7.45 (m,4H), 7.32 (t, 2H), 7.17 (t, 2H), 5.51 (d, 1H), 5.17 (m, 1H), 4.82-4.66(d of an AB quartet, 2H) 1.95, (s, 3H). ¹³C NMR (CDCl₃) δ ppm 172.87,166.17, 166.08, 165.58, 134.06, 133.91, 133.72, 130.09, 129.85, 129.80,129.37, 128.78, 128.60, 128.49, 127.96, 127.89, 79.67, 75.49, 72.60,63.29, 23.80. TOF MS ES+ (M+1: 475).

Step 3: Synthesis of scheme 5, compound 11:2,3,5-Tri-O-benzoyl-2-C-methyl-β-D-ribofuranose:

A solution of Red-Al (65 wt. % in toluene, 2.0 mL, 6.56 mmol.) inanhydrous toluene (2.0 mL) was stirred at 0° C. under argon atmosphere.A solution of anhydrous ethanol (0.38 mL, 6.56 mmol.) in anhydroustoluene (1.6 mL) was added to the toluene solution over a period of fiveminutes. The resulting mixture was stirred at 0° C. for fifteen minutesand 2 mL (2.18 mmol.) of this Red-Al/ethanol reagent was added to a cold(−5° C.) solution of 2,3,5-tri-O-benzoyl-2-C-methyl-D-ribonolactone (475mg, 1.0 mmol.) in anhydrous toluene (10 mL) over a period of 10 minutes.The reaction mixture was stirred at −5° C. for forty minutes. TLCanalysis (silica plates, 35% ethyl acetate in heptane) indicatedcomplete consumption of starting material. HPLC analysis indicated only0.1% of starting material remaining. The reaction was quenched withacetone (0.2 mL), water (15 mL) and 1 N HCl (15 mL) at 0° C. and allowedto warm to room temperature. 1 N HCl (5 mL) was added to dissolve theinorganic salts (pH: 2-3). The mixture was extracted with ethyl acetate(3×25 mL) and the organic solution washed with brine (25 mL), dried(anhydrous sodium sulfate, 10 g) and solvent removed under reducedpressure and at temperature of 40° C. to give the desired product 11 inquantitative yield (480 mg). This material was used as is for thesubsequent step.

Step 4: Synthesis of Scheme 5, Compound 12:1,2,3,5-tetra-O-benzoyl-2-C-methyl-β-D-ribofuranose:

Benzoyl chloride (283 mg, 2.0 mmol.) was added, over a period of fiveminutes, to a cold solution (5° C.) of compound 11 (480 mg, 1.0 mmol.),4-dimethylaminopyridine (12.3 mg, 0.1 mmol.) and triethylamine (506 mg,5.0 mmol.) in anhydrous tetrahydrofuran (5 mL). The reaction mixture wasstirred at room temperature and under argon atmosphere overnight. HPLCanalysis indicated 0.25% of un-reacted starting material. The reactionwas quenched by adding ice-cold water (10 g) and saturated aqueoussolution of sodium bicarbonate. Tetrahydrofuran was removed underreduced pressure and the mixture was extracted with ethyl acetate (50mL). The organic solution was washed with water (25 mL), brine (25 mL),dried (anhydrous sodium sulfate, 12 g) and solvent removed under reducedpressure to give 650 mg of thick oily product. This crude product wasstirred with 5 mL of tert-butyl methyl ether for 5 minutes and heptane(5 mL) and water (0.1 mL) were added and stirring was continued for anadditional period of two hours at 20° C. Solids were collected by vacuumfiltration and filter caked washed with 1:1 heptane:tert-butyl methylether solution (6 mL) and tert-butyl methyl ether (2 mL). Drying thesolid in vacuum gave 300 mg (52%) of desired product 12 (98.43% pure byHPLC/AUC) as a white solid that melted at 154-156.3° C. (literaturemelting point: 155-156° C.). ¹H NMR (CDCl₃) δ ppm 8.13 (m, 4H), 8.07 (d,2H), 7.89 (d, 2H), 7.63 (m, 3H), 7.48 (m, 6H), 7.15 (m, 3H), 7.06 (s,1H), 5.86 (dd, 1H), 4.79 (m, 1H), 4.70-4.52 (d of an AB quartet, 2H),1.95, (s, 3H). ¹³C NMR (CDCl₃) δ ppm 166.31, 165.83, 165.01, 164.77,134.01, 133.86, 133.70, 133.17, 130.44, 130.13, 129.97, 129.81, 129.59,129.39, 129.07, 128.84, 128.76, 128.37, 98.01, 86.87, 78.77, 76.35,64.05, 17.07. (C₃₄H₂₈O₉: calcd C, 70.34; H, 4.86. Found: C, 70.20; H,4.95).

Step 5: Synthesis of Scheme 6, Compound 13:4-Amino-1-(3,4-dibenzoyloxy-5-benzyloxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2-one

Cytosine (89 g, 0.80 mol) was suspended in acetonitrile (900 ml) in a 12L round bottomed flask equipped with a reflux condenser, overheadstirrer and an argon inlet adapter. The suspension was stirred at 20° C.under argon atmosphere and N,O-bis(trimethylsilyl)acetamide (537 ml, 2.2mol) was added in one portion. The resulting solution was heated to 80°C. and stirred for an additional hour at the same temperature.1,2,3,5-tetra-O-benzoyl-2-C-methyl-□-D-ribofuranose (425.0 g, 0.73 mol)was suspended in acetonitrile (4000 ml) and added to the reactionmixture. The reaction mixture became clear after a few minutes and thetemperature dropped to ca. 50° C. Tin(IV) chloride (154 ml, 1.31 mol)was added over a period of 15 minutes and stirring was continued at 80°C. After one hour, an aliquot of reaction mixture was quenched by addingaqueous sodium bicarbonate solution and extracting the aqueous layerwith ethyl acetate. The ethyl acetate layer was examined by TLC (silicagel, 20% ethyl acetate in heptane, R_(f) for sugar derivative: 0.40).TLC analysis indicated the complete consumption of the sugar derivative.Desired product was detected by TLC using 10% methanol indichloromethane (R_(f): 0.37). The reaction was also monitored by HPLC(Method # 2). Reaction mixture was cooled to 20° C. and quenched byadding saturated aqueous sodium bicarbonate solution (3000 ml) over aperiod of 30 minutes (observed an exotherm when added the first fewdrops of the sodium bicarbonate solution). Solid sodium bicarbonate(1350 g) was added in portions to avoid foaming. The mixture was checkedto make sure that its pH is ≧7. Agitation was stopped and layers wereallowed to separate for 20 minutes. The aqueous layer was drained andstirred with ethyl acetate (1500 ml) and the mixture was allowed toseparate (30 minutes). The organic layer was isolated and combined withthe acetonitrile solution. The organic solution was washed with brine(500 ml) and then solvent stripped to a volume of ca. 750 ml. Productcan be used as is in the subsequent reaction. It may also be furtherstripped to white foamy solid, in quantitative yield. Structure ofcompound 13 was confirmed by ¹H NMR analysis.

Step 6: Synthesis of Scheme 6, Compound mCyd:4-Amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2-one

Sodium methoxide (13.8 g, 0.26 mol) was added to a solution of compound10 (416 g, 0.73 mol) in methanol (2000 ml). The reaction mixture wasstirred at room temperature and monitored by TLC (silica gel, 10%methanol in dichloromethane, R_(f) of compound 9: 0.53) and (silica gel,30% methanol in dichloromethane, R_(f) of compound 11: 0.21). Productstarted to precipitate after 30 minutes and TLC indicated reactioncompletion after two hours. The reaction was also monitored by HPLC(Method # 2). Methanol was removed under reduced pressure to a volume ofca. 500 ml chased with ethanol (2×500 ml) to a volume of ca. 500 ml. Theresidual thick slurry was diluted with 750 ml of ethanol and the mixturewas stirred at 20° C. for one hour. Product was collected by filtration,filter cake washed with ethanol (100 ml) and tert-butyl-methyl ether(100 ml) and dried to give 168 g (90% yield for the two steps) ofproduct 11 in purity of >97% (HPLC/AUC). Product was also analyzed by ¹Hand ¹³C NMR.

Step 7: Synthesis of Scheme 6, Compound 14:2-Tert-butoxycarbonylamino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidin-1-yl)-4-hydroxy-2-hydroxymethyl-4-methyl-tetrahydro-furan-3-ylester

A solution of N-(tert-butoxycarbonyl)-L-valine (46.50 g, 214 mmol.),carbonyldiimidazole (34.70 g, 214 mmol.), and anhydrous tetrahydrofuran(1000 mL) in a 2 L round bottom flask, was stirred at 25° C. under argonfor 1.5 hours and then at 40-50° C. for 20 minutes. In a separate 5 L5-necked round bottom flask, equipped with an overhead stirrer, coolingtower, temperature probe, addition funnel, and an argon line was added4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2-one(50.0 g, 195 mmol.) and anhydrous N,N-dimethylformamide (1000 mL). Thismixture was heated at 100° C. for 20 minutes until all of thepyrimidine-2-one derivative compound went into solution, and thentriethyl amine (500 mL) and 4-dimethylaminopyridine (2.38 g, 19 mmol)were added to the solution. The mixture was next heated at 97° C. for 20minutes and the tetrahydrofuran solution was added slowly through anaddition funnel over a period of 2 hours, maintaining the temperature nolower than 82° C. The reaction mixture was heated at 82° C. for 1 hourand monitored by HPLC (product=68%, SM=11%, and impurity at about 12min=17%, excluding dimethylaminopyridine). The reaction mixture wascooled to room temperature and then triethylamine and tetrahydrofuranwere removed under vacuum at 30° C. The solution was then neutralizedwith acetic acid to a pH of 7.69. N,N-dimethylformamidine was removedunder vacuum at 35° C. and chased with ethyl acetate (2×200 mL). Thecrude product was stirred with ethyl acetate (500 mL) and water (300mL). The two layers were separated and the aqueous layer was extractedwith ethyl acetate (500 mL). The combined organic layers were washedwith an aqueous saturated brine solution (500 mL). Next the organiclayer was extracted with an aqueous solution of malonic acid (4×400 mL,10 wt. %). The organic layer was checked by TLC (silica, 20% methanol indichloromethane) to make sure that all the desired product was removedfrom the organic layer. The acidic aqueous extracts were combined andcooled in an ice bath and neutralized with triethylamine to a pH of 7.40so that the solids fell out of solution. Ethyl acetate then was added tothe aqueous layer. The white solids were collected by vacuum filtration.Drying the obtained solids in vacuum gave 81.08 g of 99.01 pure (HPLC)first crop.

Step 8: Synthesis of scheme 6, val-mCyd-2-Amino-3-methyl-butyric acid5-(4-amino-2-oxo-2H-pyrimidine-1-yl)-4-hydroxy-2hydroxy-methyl-4-methyl-tetrahydro-furan-3-yl ester (dihydrochloridesalt)

A solution of compound 14 (21.0 g, 0.046 mol) in ethanol (168 ml) wasstirred in a round bottomed flask equipped with an overhead stirrer,temperature probe, argon line and hydrogen chloride gas bubbler.Hydrogen chloride gas (22 g) was bubbled into the clear solution over aperiod of one hour. The reaction temperature was kept below 30° C. usingan ice-water bath. Solid formation started after a few minutes ofintroducing the hydrogen chloride gas. After 4 hours, HPLC (method # 3)showed only 0.8% of starting material. Solids were collected byfiltration and filter cake washed with ethanol (20 ml) and di-ethylether (100 ml). After drying product under vacuum for 16 hours, 19.06 g(96.5%) of val-mCyd was obtained in 97.26% purity (HPLC, method # 3);m.p. 210° C. (brown), 248-250° C. (melted); ¹H NMR (DMSO-d₆) δ ppm 10.0(s, 1H, ½NH₂, D₂O exchangeable), 8.9-8.6 (2 br s, 4H, ½NH₂, NH₃, D₂Oexchangeable), 8.42 (d, 1H, H-6, J 5-6=7.9 Hz), 6.24 (d, 1H, H-5,J₅₋₆=7.9 Hz), 5.84 (s, 1H, H-1′), 5.12 (d, 1H, H-3′, J_(3′-4′)=8.8 Hz),4.22 (d, 1H, H-4, J_(3′-4′)=8.7 Hz), 4.0-3.9 (m, 1H, CH), 3.8-3.5 (m,2H, H-5′, H-5″), 2.3-2.1 (m, 1H, CH), 1.16 (s, 3H, CH₃), 1.0 (m, 6H,(CH₃)₂CH); FAB>0 (GT) 713 (2M+H)⁺, 449 (M+G+H)⁺, 357 (M+H)⁺, 246 (S)⁺,112 (B+2H)⁺; FAB<0 (GT) 747 (2M+Cl)⁻, 483 (M+G+Cl)⁻, 391 (M+Cl)⁻, 355(M−H)⁻, 116 (Val)⁻, 110 (B)⁻, 35 (Cl)⁻.

Two different HPLC methods were used to analyze the above compounds.Both methods use the following reverse phase column. In method 1, thecolumn was run at a flow rate of 1.00 ml/min of an acetonitrile/waterlinear gradient for a 20 minute run time. Five-minute equilibration wasallowed between runs. The measurements were at 254 nm. TABLE A Retentiontime of key intermediates: Scheme 5, Compound Retention Time Compund 1010.2 min Compund 11  9.4 min Compund 12 12.9 min

In the second method, identification was determined at 272 nm. A WatersNovapak C18, 3.9×150 mm ID, 4 μm particle size, 60 Å pore size orequivalent can be used. The chromatographic conditions are as follows:injection volume=10 μl, column temperature=25° C., flow rate=1.00ml/min, ultraviolet detector at 272 nm, run time is 35 minutes. Thesystem suitability requirement for the percent relative standarddeviation for the reference standard is not more than 1.0%. TABLE BPurity and impurities are determined at 272 nm Solvent A - 20 nM SolventB - triethylammonium Acetonitrile, Time (minutes) acetate buffer HPLCgrade. 0.00 100.0 0.0 10.00 85.0 15.0 25.00 5.0 95.0 35.0 5.0 95.0

TABLE C Retention times of key intermediates and final drug substanceScheme 6, Compound Retention Time (minutes) Compound mCyd 2.7-2.8Compund 14 15.5 val-mCyd 9.1

A process of synthesizing aβ-D-2′-C-methyl-2′-acetyl-ribofuransyl-cytidine-3′-O-L-valine ester isdetailed in FIG. 7. A process of synthesizing aβ-D-2′-C-methyl-2′-acetyl-ribofuransyl-cytidine-3′-O-L-proline ester isdetailed in FIG. 8. A process for synthesizing aβ-D-2′-C-methyl-2′-acetyl-ribofuransyl-cytidine-3′-O-L-alanine ester isdepicted in FIG. 9. A process of synthesizing aβ-D-2′-C-methyl-2′-(cyclohexanecarboxylate)-ribofuransyl-cytidine-3′-O-L-valine ester is depicted inFIG. 10. These processes can be accomplished using techniques similar tothose described above.

C. General Synthesis of 3′-C-Branched Nucleosides

3′-C-Branched ribonucleosides of the following structure:

wherein BASE is a purine or pyrimidine base as defined herein;R⁷ and R⁹ are independently hydrogen, OR², hydroxy, alkyl (includinglower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), chlorine, bromine, iodine, NO₂, NH₂, —NH(loweralkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;R⁸ is H, alkyl (including lower alkyl), chlorine, bromine or iodine;alternatively, R⁷ and R⁹, or R⁸ and R⁹ can come together to form a pibond;R¹ and R² are independently H; phosphate (including monophosphate,diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl(including lower acyl); alkyl (including lower alkyl); sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein; alipid, including a phospholipid; an amino acid; a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R¹ or R² is independently H or phosphate;R⁶ is an alkyl, chloro-, fluoro-, bromo-, iodo-alkyl (i.e. CF₃),alkenyl, or alkynyl (i.e. allyl); andX is O, S, SO₂ or CH₂can be prepared by one of the following general methods.1. Glycosylation of the Nucleobase with an Appropriately Modified Sugar

The key starting material for this process is an appropriatelysubstituted sugar with a 3′-OH and 3′-H, with the appropriate leavinggroup (LG), for example an acyl group or a chloro, bromo, fluoro, iodo.The sugar can be purchased or can be prepared by any known meansincluding standard epimerization, substitution, oxidation and reductiontechniques. The substituted sugar can then be oxidized with theappropriate oxidizing agent in a compatible solvent at a suitabletemperature to yield the 3′-modified sugar. Possible oxidizing agentsare Jones reagent (a mixture of chromic acid and sulfuric acid),Collins's reagent (dipyridine Cr(VI) oxide, Corey's reagent (pyridiniumchlorochromate), pyridinium dichromate, acid dichromate, potassiumpermanganate, MnO₂, ruthenium tetroxide, phase transfer catalysts suchas chromic acid or permanganate supported on a polymer, Cl₂-pyridine,H₂O₂-ammonium molybdate, NaBrO₂-CAN, NaOCl in HOAc, copper chromite,copper oxide, Raney nickel, palladium acetate, Meerwin-Pondorf-Verleyreagent (aluminum t-butoxide with another ketone) andN-bromosuccinimide.

Then coupling of an organometallic carbon nucleophile, such as aGrignard reagent, an organolithium, lithium dialkylcopper or R⁶—SiMe₃ inTBAF with the ketone with the appropriate non-protic solvent at asuitable temperature, yields the 3′-C-branched sugar. The 3′-C-branchedsugar can be optionally protected with a suitable protecting group,preferably with an acyl or silyl group, by methods well known to thoseskilled in the art, as taught by Greene et al. Protective Groups inOrganic Synthesis, John Wiley and Sons, Second Edition, 1991.

The optionally protected sugar can then be coupled to the BASE bymethods well known to those skilled in the art, as taught by TownsendChemistry of Nucleosides and Nucleotides, Plenum Press, 1994. Forexample, an acylated sugar can be coupled to a silylated base with aLewis acid, such as tin tetrachloride, titanium tetrachloride ortrimethylsilyltriflate in the appropriate solvent at a suitabletemperature. Alternatively, a halo-sugar can be coupled to a silylatedbase with the presence of trimethylsilyltriflate.

Subsequently, the nucleoside can be deprotected by methods well known tothose skilled in the art, as taught by Greene et al. Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991.

In a particular embodiment, the 3′-C-branched ribonucleoside is desired.The synthesis of a ribonucleoside is shown in Scheme 7. Alternatively,deoxyribo-nucleoside is desired. To obtain these nucleosides, the formedribonucleoside can optionally be protected by methods well known tothose skilled in the art, as taught by Greene et al. Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991, andthen the 2′-OH can be reduced with a suitable reducing agent.Optionally, the 2′-hydroxyl can be activated to facilitate reduction;i.e. via the Barton reduction.

2. Modification of a Preformed Nucleoside

The key starting material for this process is an appropriatelysubstituted nucleoside with a 3′-OH and 3′-H. The nucleoside can bepurchased or can be prepared by any known means including standardcoupling techniques. The nucleoside can be optionally protected withsuitable protecting groups, preferably with acyl or silyl groups, bymethods well known to those skilled in the art, as taught by Greene etal. Protective Groups in Organic Synthesis, John Wiley and Sons, SecondEdition, 1991.

The appropriately protected nucleoside can then be oxidized with theappropriate oxidizing agent in a compatible solvent at a suitabletemperature to yield the 2′-modified sugar. Possible oxidizing agentsare Jones reagent (a mixture of chromic acid and sulfuric acid),Collins's reagent (dipyridine Cr(VI) oxide, Corey's reagent (pyridiniumchlorochromate), pyridinium dichromate, acid dichromate, potassiumpermanganate, MnO₂, ruthenium tetroxide, phase transfer catalysts suchas chromic acid or permanganate supported on a polymer, Cl₂-pyridine,H₂O₂-ammonium molybdate, NaBrO₂-CAN, NaOCl in HOAc, copper chromite,copper oxide, Raney nickel, palladium acetate, Meerwin-Pondorf-Verleyreagent (aluminum t-butoxide with another ketone) andN-bromosuccinimide.

Subsequently, the nucleoside can be deprotected by methods well known tothose skilled in the art, as taught by GreeneGreene et al. ProtectiveGroups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991.

In a particular embodiment, the 3′-C-branched ribonucleoside is desired.The synthesis of a ribonucleoside is shown in Scheme 8. Alternatively,deoxyribo-nucleoside is desired. To obtain these nucleosides, the formedribonucleoside can optionally be protected by methods well known tothose skilled in the art, as taught by Greene et al. Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991, andthen the 2′-OH can be reduced with a suitable reducing agent.Optionally, the 2′-hydroxyl can be activated to facilitate reduction;i.e. via the Barton reduction.

In another embodiment of the invention, the L-enantiomers are desired.Therefore, the L-enantiomers can be corresponding to the compounds ofthe invention can be prepared following the same foregoing generalmethods, beginning with the corresponding L-sugar or nucleosideL-enantiomer as starting material.

D. General Synthesis of 4′-C-Branched Nucleosides

4′-C-Branched ribonucleosides of the following structure:

wherein BASE is a purine or pyrimidine base as defined herein;R⁷ and R⁹ are independently hydrogen, OR², hydroxy, alkyl (includinglower alkyl), azido, cyano, alkenyl, alkynyl, Br-vinyl, —C(O)O(alkyl),—C(O)O(lower alkyl), —O(acyl), —O(lower acyl), —O(alkyl), —O(loweralkyl), —O(alkenyl), chlorine, bromine, iodine, NO₂, NH₂, —NH(loweralkyl), —NH(acyl), —N(lower alkyl)₂, —N(acyl)₂;R⁸ and R¹⁰ are independently H, alkyl (including lower alkyl), chlorine,bromine or iodine; alternatively, R⁷ and R⁹, R⁷ and R¹⁰, R⁸ and R⁹, orR⁵ and R¹⁰ can come together to form a pi bond;R¹ and R² are independently H; phosphate (including monophosphate,diphosphate, triphosphate, or a stabilized phosphate prodrug); acyl(including lower acyl); alkyl (including lower alkyl); sulfonate esterincluding alkyl or arylalkyl sulfonyl including methanesulfonyl andbenzyl, wherein the phenyl group is optionally substituted with one ormore substituents as described in the definition of aryl given herein; alipid, including a phospholipid; an amino acid; a carbohydrate; apeptide; cholesterol; or other pharmaceutically acceptable leaving groupwhich when administered in vivo is capable of providing a compoundwherein R¹ is independently H or phosphate;R⁶ is an alkyl, halogeno-alkyl (i.e. CF₃), alkenyl, or alkynyl (i.e.allyl); and

X is O, S, SO₂ or CH₂

can be prepared by the following general method.

Modification from the Pentodialdo-Furanose

The key starting material for this process is an appropriatelysubstituted pentodialdo-furanose. The pentodialdo-furanose can bepurchased or can be prepared by any known means including standardepimerization, substitution and cyclization techniques.

In a preferred embodiment, the pentodialdo-furanose is prepared from theappropriately substituted hexose. The hexose can be purchased or can beprepared by any known means including standard epimerization (e.g. viaalkaline treatment), substitution and coupling techniques. The hexosecan be either in the furanose form, or cyclized via any means known inthe art, such as methodology taught by Townsend Chemistry of Nucleosidesand Nucleotides, Plenum Press, 1994, preferably by selectivelyprotecting the hexose, to give the appropriate hexafuranose.

The 4′-hydroxymethylene of the hexafuranose then can be oxidized withthe appropriate oxidizing agent in a compatible solvent at a suitabletemperature to yield the 4′-aldo-modified sugar. Possible oxidizingagents are Swern reagents, Jones reagent (a mixture of chromic acid andsulfuric acid), Collins's reagent (dipyridine Cr(VI) oxide, Corey'sreagent (pyridinium chlorochromate), pyridinium dichromate, aciddichromate, potassium permanganate, MnO₂, ruthenium tetroxide, phasetransfer catalysts such as chromic acid or permanganate supported on apolymer, Cl₂-pyridine, H₂O₂-ammonium molybdate, NaBrO₂-CAN, NaOCl inHOAc, copper chromite, copper oxide, Raney nickel, palladium acetate,Meerwin-Pondorf-Verley reagent (aluminum t-butoxide with another ketone)and N-bromosuccinimide, though preferably using H₃PO₄, DMSO and DCC in amixture of benzene/pyridine at room temperature.

Then, the pentodialdo-furanose can be optionally protected with asuitable protecting group, preferably with an acyl or silyl group, bymethods well known to those skilled in the art, as taught by Greene etal. Protective Groups in Organic Synthesis, John Wiley and Sons, SecondEdition, 1991. In the presence of a base, such as sodium hydroxide, theprotected pentodialdo-furanose can then be coupled with a suitableelectrophilic alkyl, halogeno-alkyl (i.e. CF₃), alkenyl or alkynyl (i.e.allyl), to obtain the 4′-alkylated sugar. Alternatively, the protectedpentodialdo-furanose can be coupled with the corresponding carbonyl,such as formaldehyde, in the presence of a base, such as sodiumhydroxide, with the appropriate polar solvent, such as dioxane, at asuitable temperature, which can then be reduced with an appropriatereducing agent to give the 4′-alkylated sugar. In one embodiment, thereduction is carried out using PhOC(S)Cl, DMAP, preferably inacetonitrile at room temperature, followed by treatment of ACCN and TMSSrefluxed in toluene.

The optionally activated sugar can then be coupled to the BASE bymethods well known to those skilled in the art, as taught by TownsendChemistry of Nucleosides and Nucleotides, Plenum Press, 1994. Forexample, an acylated sugar can be coupled to a silylated base with aLewis acid, such as tin tetrachloride, titanium tetrachloride ortrimethylsilyltriflate in the appropriate solvent at a suitabletemperature.

Subsequently, the nucleoside can be deprotected by methods well known tothose skilled in the art, as taught by Greene et al. Protective Groupsin Organic Synthesis, John Wiley and Sons, Second Edition, 1991.

In a particular embodiment, the 4′-C-branched ribonucleoside is desired.Alternatively, deoxyribo-nucleoside is desired. To obtain thesedeoxyribo-nucleosides, a formed ribo-nucleoside can optionally beprotected by methods well known to those skilled in the art, as taughtby Greene et al. Protective Groups in Organic Synthesis, John Wiley andSons, Second Edition, 1991, and then the 2′-OH can be reduced with asuitable reducing agent. Optionally, the 2′-hydroxyl can be activated tofacilitate reduction; i.e. via the Barton reduction.

In another embodiment of the invention, the L-enantiomers are desired.Therefore, the L-enantiomers can be corresponding to the compounds ofthe invention can be prepared following the same foregoing generalmethods, beginning with the corresponding L-pentodialdo-furanose asstarting material.

E. General Synthesis of 2′ and/or 3′-Prodrugs

The key starting material for this process is an appropriatelysubstituted 1′, 2′, 3′ or 4′-branched β-D or β-L nucleosides. Thebranched nucleoside can be purchased or can be prepared by any knownmeans including the techniques disclosed herein. The branched nucleosidecan be optionally protected with a suitable protecting group, preferablywith a silyl group, by methods well known to those skilled in the art,as taught by Greene et al. Protective Groups in Organic Synthesis, JohnWiley and Sons, Second Edition, 1991. The optionally protected branchednucleoside can then be coupled with a suitable acyl doner, such as anacyl chloride and/or an acyl anhydride with the appropriate protic oraprotic solvent at a suitable temperature, to give the 2′ and/or 3′prodrug of 1′, 2′, 3′ or 4′-branched β-D or β-L nucleoside. (SyntheticCommunications, 1978, 8(5), 327-333; J. Am. Chem. Soc., 1999, 121(24),5661-5664.) Alternatively, the optionally protected branched nucleosidecan then be coupled with a suitable acyl, such as a carboxylic acid,such as alkanoic acid and/or amino acid residue, optionally with asuitable coupling agent, with the appropriate aprotic solvent at asuitable temperature, to give the 2′ and/or 3′ prodrug of 1′, 2′, 3′ or4′-branched β-D or β-L nucleoside. Possible coupling reagents are anyreagents that promote coupling, including but are not limiting to,Mitsunobu reagents (e.g. diisopropyl azodicarboxylate and diethylazodicarboxylate) with triphenylphosphine or various carbodiimides. Inone embodiment, for a 3′-prodrug of a 2′-branched nucleoside, thenucleoside is preferably not protected and is directly coupled to analkanoic acid or amino acid residue with an appropriate couplingreagient, such as a carbodiimide.

For example, simple amino-alcohols can be esterified using acidchlorides in refluxing acetonitrile-benzene mixture (See Scheme 9 below:Synthetic Communications, 1978, 8(5), 327-333; hereby incorporated byreference). Alternatively, esterification can be achieved using ananhydride, as described in J. Am. Chem. Soc., 1999, 121(24), 5661-5664,which is hereby incorporated by reference. See FIGS. 2, 3 and 4.

The present invention is described by way of illustration, in thefollowing examples. It will be understood by one of ordinary skill inthe art that these examples are in no way limiting and that variationsof detail can be made without departing from the spirit and scope of thepresent invention.

EXAMPLE 1 Preparation of 1′-C-methylriboadenine via6-amino-9-(1-deoxy-β-D-psicofuranosyl)purine

Melting points were determined on a Mel-temp II apparatus and areuncorrected. NMR spectra were recorded on a Bruker 400 AMX spectrometerat 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR with TMS as internalstandard. Chemical shifts (6) are reported in parts per million (ppm),and signals are reported as s (singlet), d (doublet), t (triplet), q(quartet), m (multiplet), or bs (broad singlet). IR spectra weremeasured on a Nicolet 510P FT-IR spectrometer. Mass spectra wererecorded on a Micromass Autospec high-resolution mass spectrometer. TLCwere performed on Uniplates (silica gel) purchased from Analtech Co.Column chromatography was performed using either silica gel-60 (220-440mesh) for flash chromatography or silica gel G (TLC grade, >440 mesh)for vacuum flash column chromatography. UV spectra were obtained on aBeckman DU 650 spectrophotometer. Elemental analysis was performed byAtlantic Microlab, Inc., Norcross, Ga., or Galbraith Laboratories, Inc.,Knoxville, Tenn. HPLC was performed with a Waters HPLC system (MilliporeCorporation, Milford, Mass.) equipped with a Model 600 controller, aModel 996 photodiode array detector and a Model 717 plus autosampler.Millennium 2010 software was used for system control, data acquisitionand processing. A chiralyser polarimetric detector, Perkin-Elmer Model241MC polarimeter (Wilton, Conn.), was used for the determination ofoptical rotations.

The title compound can be prepared according to a published procedure(J. Farkas, and F. Sorm, “Nucleic acid components and their analogues.XCIV. Synthesis of 6-amino-9-(1-deoxy-β-D-psicofuranosyl)purine”,Collect. Czech. Chem. Commun. 1967, 32, 2663-2667. J. Farkas”, Collect.Czech. Chem. Commun. 1966, 31, 1535) (Scheme 10).

In a similar manner, but using the appropriate sugar and pyrimidine orpurine bases, the following nucleosides of Formula I are prepared.

wherein R¹, R², R³, X¹, X², and Y are defined in Table 1.Alternatively, the following nucleosides of Formula IV are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R³, X¹, Y are defined in Table 2.Alternatively, the following nucleosides of Formula VII are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R³, R⁶, X, and Base are defined in Table 3.

Alternatively, the following nucleosides of Formula VIII are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R⁶, X, and Base are defined in Table 4.Alternatively, the following nucleosides of Formula XXI are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R⁶, X and Base are defined in Table 5.

Alternatively, the following nucleosides of Formula XIII are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R⁶, R⁷, R⁸, X, Base, R¹⁰ and R⁹ are defined in Table 6.

EXAMPLE 2 Preparation of 2′-C-methylriboadenine

The title compound was prepared according to a published procedure (R.E. Harry-O'kuru, J. M. Smith, and M. S. Wolfe, “A short, flexible routetoward 2′-C-branched ribonucleosides”, J. Org. Chem. 1997, 62,1754-1759) (Scheme 11).

The 3′-prodrug of the 2′-branched nucleoside was prepared according topublished procedure (Synthetic Communications, 1978, 8(5), 327-333; J.Am. Chem. Soc., 1999, 121(24), 5661-5664). Alternatively, the2′-branched nucleoside can be esterified without protection (Scheme11b). Carbonyldiimidazole (377 mg, 2.33 mmol) was added to a solution ofN-(tert-butoxycarbonyl)-L-valine (507 mg, 2.33 mmol) in 15 mL ofanhydrous tetrahydrofuran. The mixture was stirred at 20° C. for onehour and at 50° C. for 10 minutes and then added to a solution of4-Amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2-one(500 mg, 1.95 mmol), 4-(dimethylamino)pyridine (25 mg, 0.195 mmol),triethylamine (5 mL) in anhydrous N,N-dimethylformamide (10 mL), whichis also stirring at 50° C. The reaction mixture was stirred at 50° C.for one hour and then examined by HPLC*. HPLC analysis indicated theformation of 52% of the desired ester, 17% of starting material inaddition to undesired by-products. The 3′-OH of4-amino-1-(3,4-dihydroxy-5-hydroxymethyl-3-methyl-tetrahydro-furan-2-yl)-1H-pyrimidine-2-onetends to react selectively when coupled with BOC-Val.

The product was analyzed by HPLC using a reverse phase column: Waterspart # WAT086344; Nova-Pak C18, 60 Å pore size, 4 μm particle size,3.9×150 mm. Chromatograms were generated using a Waters 2695 HPLC and996 PDA detector. Mobile Phase: HPLC grade acetonitrile and water werebought from JT Baker and 1M solution of triethylammonium acetate fromFluka.

Flow rate: 1.00 mL/min. of an acetonitrile/20 mM aqueoustriethylammonium acetate buffer gradient as described below.

System is equilibrated for five minutes between runs.

Wave length: 272 nm. TABLE D Column Specifications Time % Acetonitrile %Buffer 0.00 0.00 100.0 15.00 80.0 20.0 30.00 80.0 20.0

TABLE E Description of compounds vs. retention times: Compound RETENTIONTIME (IN MINUTES) Desired ester 8.3 DMAP 3.7 (Broad Peak) Startingmaterial 2.7

In a similar manner, but using the appropriate sugar and pyrimidine orpurine bases, the following nucleosides of Formula II are prepared.

wherein R², R³, X¹, X², and Y are defined in Table 7.

Alternatively, the following nucleosides of Formula V are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R¹, X¹ and Y are defined in Table 8.

Alternatively, the following nucleosides of Formula IX are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R³, R⁶, X, and Base are defined in Table 9.

Alternatively, the following nucleosides of Formula X are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R⁷, R⁶, X, and Base are defined in Table 10.

Alternatively, the following nucleosides of Formula XXII are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R⁶, X, and Base are defined in Table 11.

Alternatively, the following nucleosides of Formula XIII are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R⁶, R⁷, X, Base, R⁹ and R¹⁰ are defined in Table 12.

EXAMPLE 3 Preparation of 3′-C-methylriboadenine

The title compound can be prepared according to a published procedure(R. F. Nutt, M. J. Dickinson, F. W. Holly, and E. Walton,“Branched-chain sugar nucleosides. III. 3′-C-methyladenine”, J. Org.Chem. 1968, 33, 1789-1795) (Scheme 12).

In a similar manner, but using the appropriate sugar and pyrimidine orpurine bases, the following nucleosides of Formula III are prepared.

wherein R¹, R², R³, X¹, X², and Y are defined in Table 13.

Alternatively, the following nucleosides of Formula VI are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R³, X¹, and Y are defined in Table 14.

Alternatively, the following nucleosides of Formula XI are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R³, R⁶, X, and Base are defined in Table 15.

Alternatively, the following nucleosides of Formula XII are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R⁶, X and Base are defined in Table 16.

Alternatively, the following nucleosides of Formula XXIII are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R⁶, X and Base are defined in Table 17.

Alternatively, the following nucleosides of Formula XV are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R⁶, R⁷, X, Base, R⁸ and R⁹ are defined in Table 18.

EXAMPLE 4 Preparation of1-O-methyl-2,3-O-isopropylidene-β-D-ribofuranose (AA)

The title compound can be prepared according to a published procedure(Leonard, N.J.; Carraway, K. L. “5-Amino-5-deoxyribose derivatives.Synthesis and use in the preparation of “reversed” nucleosides” J.Heterocycl. Chem. 1966, 3, 485-489).

A solution of 50.0 g (0.34 mole) of dry D-ribose in 1.0 L of acetone,100 mL of 2,2-dimethoxypropane, 200 mL of methanol containing 20 mL ofmethanol saturated with hydrogen chloride at 0° C. was stirred overnightat room temperature. The resulting solution was neutralized withpyridine and evaporated under reduced pressure. The resulting oil waspartitioned between 400 mL of water and 400 mL of methylene chloride.The water layer was extracted twice with methylene chloride (400 mL).The combined organic extracts were dried over sodium sulfate andevaporated under reduced pressure. The residue was purified by silicagel column chromatography [eluent: stepwise gradient of methanol (1-2%)in methylene chloride] to give pure AA (52.1 g, 75%) as a yellow syrup.¹H-NMR (CDCl₃): δ 5.00 (s, 1H, H-1), 4.86 (d, 1H, H-2, J₂₋₃=5.9 Hz),4.61 (d, 1H, H-3, J₃₋₂=5.9 Hz), 4.46 (t, 1H, H-4, J₄₋₅=2.7 Hz),3.77-3.61 (m, 2H, H-5 and H-5′), 3.46 (s, 1H, OCH₃), 3.0-2.4 (br s, 1H,OH-5), 1.51 (s, 3H C₃), 1.34 (s, 3H C₃); MS (matrix GT): FAB>0 m/z 173(M-OCH3)⁺.

EXAMPLE 5 Preparation of1-O-methyl-2,3-O-isopropylidene-β-D-pentodialdo-ribofuranose (BB)

The title compound can be prepared according to a published procedure(Jones, G. H.; Moffatt, J. G. Oxidation of carbohydrates by thesulfoxide-carbodiimide and related methods. Oxidation withdicyclohexylcarbodiimide-DMSO, diisopropylcarbodiimide-DMSO, aceticanhydride-DMSO, and phosphorus pentoxide-DMSO: in Methods inCarbohydrate Chemistry; Whisler, R. L. and Moffatt, J. L. Eds; AcademicPress: New York, 1972; 315-322).

Compound AA was co-evaporated twice with anhydrous pyridine.Dicyclohexylcarbodi-imide (DCC, 137.8 g, 0.67 mol) was added to asolution of AA (68.2 g, 0.33 mole) in anhydrous benzene (670 mL), DMSO(500 mL) and pyridine (13.4 mL). To the resulting solution, cooled to 0°C., was added a solution of anhydrous crystalline orthophosphoric acid(16.4 g, 0.167 mmol) in anhydrous DMSO (30 mL). The mixture was stirredfor 1.5 hours at 0° C. and 18 hours at room temperature under argonatmosphere, diluted with ethyl acetate (1000 mL). A solution of oxalicacid dihydrate (63.1 g, 038 mol) in DMSO (30 mL) was added and thereaction mixture was stirred at room temperature during 1 hour and thenfiltered to eliminate precipitated dicyclohexylurea (DCU). The filtratewas concentrated to a volume of about 600 mL under reduced pressure andneutralized with a saturated aqueous sodium hydrogen carbonate solution(400 mL). Brine (200 mL) was added and the organic layer was extractedwith ethyl acetate (4×1000 mL). The combined organic layers wereconcentrated to a volume of about 2000 mL, washed with a saturatedaqueous sodium hydrogen carbonate solution (2×700 mL), and with brine(2×700 mL) before being dried over sodium sulfate and evaporated underreduced pressure. A small fraction of the crude residue was purified onsilica gel chromatography [eluent: chloroform/ethyl ether, 8:2] in orderto confirm the structure of BB which was obtained as a pale yellowsolid. ¹H-NMR (CDCl₃): δ 9.61 (s, 1H, H-5), 5.12 (s, 1H, H-1), 5.08 (d,1H, H-2, J₂₋₃=5.9 Hz), 4.53 (d, 1H, H-3, J₃₋₂=6.0 Hz), 4.51 (s, 1H,H-4), 3.48 (s, 1H, OCH₃), 1.56 (s, 3H C₃), 1.36 (s, 3H CH₃); MS (matrixGT): FAB>0 m/z 203 (M+H)⁺, 171 (M−OCH₃)⁺.

EXAMPLE 6 Preparation of4-C-hydroxymethyl-1-O-methyl-2,3-O-isopropylidene-β-D-ribofuranose (CC)

The title compound can be prepared according to a published procedure(Leland, D. L.; Kotick, M. P. Carbohydr. Res. 1974, 38, C9-C11; Jones,G. H.; Taniguchi, M., et al. J. Org. Chem. 1979, 44, 1309-1317; Gunic,E.; Girardet, J.-L.; et al. Bioorg. Med. Chem. 2001, 9, 163-170).

To a solution of the crude material (BB) obtained above and 37% aqueousformaldehyde (167 mL) in dioxane (830 mL) was added aqueous sodiumhydroxyde (2N, 300 mL). The mixture was stirred at room temperature for4 hours and neutralized by addition of Dowex 50 W X 2 (H⁺ form). Theresin was filtered, washed with methanol, and the combined filtrateswere concentrated to dryness and coevaporated several times withabsolute ethanol. Sodium formate which was precipitated from absoluteethanol was removed by filtration, the filtrate was concentrated todryness and the residue was purified by silica gel column chromatography[eluent: stepwise gradient of methanol (0-4%) in chloroform] to givepure CC (42.2 g, 54% from AA), which was recrystallized fromcyclohexane. Mp=94-95 (dec.) (lit.94-96.5; 97-98: Refs:3,4), ¹H-NMR(DMSO-d₆): δ 4.65 (s, 1H, H-1), 4.44-4.37 (m, 3H, H-2, H-3 and OH-6),4.27 (t, 1H, OH-5, J=5.6 Hz, J=6.0 Hz), 3.42-3.34 (m, 2H, H-5 and H-6)3.29 (dd, 1H, H-5′, J_(5′-OH)=5.4 Hz, J5-5′=11.4 Hz), 3.11 (dd, 1H,H-6′, J_(6′-OH)=5.7 Hz, J6-6′=10.9 Hz), 3.03 (s, 3H, OCH₃), 1.48 (s, 3HC₃), 1.05 (s, 3H C₃); MS (matrix GT): FAB>0 m/z 469 (2M+H)⁺, 235 (M+H)⁺,203 (M-OCH₃)+FAB<0 m/z 233 (M−H)⁻.

EXAMPLE 7 Preparation of6-O-monomethoxytrityl-4-C-hydroxymethyl-1O-methyl-2,3-O-isopropylidene-β-D-ribofuranose(DD)

The title compound can be prepared according to a published procedure(Gunic, E.; Girardet, J.-L.; et al. Bioorg. Med. Chem. 2001, 9,163-170).

To a solution of CC (41.0 g, 175 mmol) in pyridine (700 ml) was added byportions dimethoxytrityl chloride (60.5 g, 178 mmol) at 4° C. Thereaction mixture was stirred for 3 hours at room temperature. Afteraddition of methanol, the reaction mixture was concentrated (200 ml) andthen dissolved with ethyl acetate (2 L). The organic layer was washedwith a 5% aqueous sodium hydrogen carbonate solution, with water anddried over sodium sulfate and then evaporated to dryness. Purificationby silica gel column chromatography [eluent: ethyl acetate/hexane 15/85]afforded pure DD (63.0 g, 68%) as a syrup. ¹H-NMR (CDCl₃): δ 7.5-6.9 (m,13H, MMTr), 4.89 (s, 1H, H-1), 4.72-4.62 (m, 3H, H-2, H-3 and OH-5),3.82 (dd, 1H, H-5, J_(5-OH)=5.5 Hz, J5-5′=10.5 Hz), 3.79 (s, 6H, OCH3),3.54 (dd, 1H, H-5′, J_(5′-OH)=4.9 Hz, J_(5′-5)=10.5 Hz), 3.31 (s, 3H,OCH₃), 3.24 (d, 1H, H-6, J_(6-6′)=9.2 Hz), 3.13 (d, 1H, H-6′,J_(6′-6)=9.2 Hz), 1.24 (s, 3H C₃), 1.15 (s, 3H C₃); MS (matrix GT):FAB>0 m/z 303 (DMTr)⁺.

EXAMPLE 8 Preparation of5-O-benzoyl-4-C-hydroxymethyl-1-O-methyl-2,3-O-isopropylidene-β-D-ribo-furanose(EE)

The title compound can be prepared according to a published procedure(Gunic, E.; Girardet, J.-L.; Pietrzkowski, Z.; Esler, C.; Wang, G.“Synthesis and cytotoxicity of 4′-C- and 5′-C-substituted Toyocamycins”Bioorg. Med. Chem. 2001, 9, 163-170).

To a solution of DD (2.51 g, 4.68 mmol) in anhydrous pyridine (37 mL)was added under argon benzoyl chloride (1.09 mL, 9.36 mmol) and thereaction mixture was stirred for 13 hours at to room temperature. Thenthe reaction was cooled to 0° C. and stopped with ice-cold water (100mL). The water layer was extracted with methylene chloride (3□ 200 mL).The combined organic layers were washed with a saturated aqueous sodiumhydrogen carbonate solution (2×150 mL), with water (1×150 mL) and thendried over sodium sulfate and evaporated under reduced pressure. Theresidue was dissolved in 80% acetic acid (70.2 mL) and the mixture wasstirred at room temperature for 3 hr and concentrated to dryness.Purification by silica gel column chromatography [eluent: chloroform]afforded pure EE (1.40 g, 88%) as a syrup. ¹H-NMR (CDCl₃): δ 8.1-7.4 (m,5H, C₆H₅CO), 5.08 (s, 1H, H-1), 4.77 (dd, 2H, H-2 and H-3, J=6.1 Hz,J=8.2 Hz), 4.51 (q, 2H, H-5 and H-5′, J=11.5 Hz, J_(5-5′)=23.8 Hz), 3.91(t, 2H, H-6 and H-6′, J=12.3 Hz), 4.38 (s, 1H, OCH₃), 2.2-1.8 (brs, 1H,OH-6), 1.57 (s, 3H C₃), 1.38 (s, 3H C₃); MS (matrix GT): FAB>0 m/z 677(2M+H)⁺, 339 (M+H)⁺, 307 (M−OCH₃)⁺, 105 (C₆H₅CO)⁺ FAB<0 m/z 121(C₆H₅CO₂)—.

EXAMPLE 9 Preparation of5-O-benzoyl-4-C-methyl-1-O-methyl-2,3-O-isopropylidene-β-D-ribofuranose(FF)

The title compound can be prepared according to a published procedure(Gunic, E.; Girardet, J.-L.; et al. Bioorg. Med. Chem. 2001, 9,163-170).

A solution of EE (37.6 g, 0.111 mol), 4-dimethylaminopyridine (DMAP,40.7 g, 0.333 mol) and phenoxythiocarbonyle chloride in anhydrousacetonitrile (1000 mL) was stirred at room temperature for 1 hour andconcentrated to dryness. The residue was dissolved in methylene chloride(500 mL) and successively washed with 0.2 M hydrochloric acid (2×500 mL)and water (500 mL) before being dried over sodium sulfate, evaporatedunder reduced pressure and coevaporated several times with anhydroustoluene. The crude material was dissolved in anhydrous toluene (880 mL)and tris(trimethylsilyl)silane (TMSS, 42.9 mL, 0.139 mol), and1,1′-azobis(cyclohexanecarbonitrile) (ACCN, 6.8 g, 27.8 mmol) wereadded. The reaction mixture was stirred under reflux for 45 minutes,cooled to room temperature and concentrated under reduced pressure. Theresulting residue was purified by silica gel column chromatography[eluent: stepwise gradient of diethyl ether (5-20%) in petroleum ether]to give pure FF (26.4 g, 74%) as a pale yellow syrup. ¹H-NMR (DMSO-d₆):δ 8.0-7.5 (m, 5H, C₆H₅CO), 4.85 (s, 1H, H-1), 4.63 (dd, 2H, H-2 and H-3,J=6.1 Hz, J=11.6 Hz), 4.24 (d, 1H, H-5, J_(5-5′)=11.1 Hz), 4.10 (d, 1H,H-5′, J_(5′-5)=11.1 Hz), 3.17 (s, 1H, OCH₃), 1.38 (s, 3H C₃), 1.30 (s,3H C₃), 1.25 (s, 3H C₃); MS (matrix GT): FAB>0 m/z 291 (M−OCH₃)⁺, 105(C₆H₅CO)⁺ FAB<0 m/z 121 (C₆H₅CO₂)⁻.

EXAMPLE 10 Preparation of5-O-benzoyl-4-C-methyl-1,2,3-O-acetyl-α,β-D-ribofuranose (GG)

Compound FF (22.5 g, 70 mmol) was suspended in a 80% aqueous acetic acidsolution (250 mL). The solution was heated at 100° C. for 3 hours. Thevolume was then reduced by half and coevaporated with absolute ethanoland pyridine. The oily residue was dissolved in pyridine (280 mL) andthen cooled at 0° C. Acetic anhydride (80 mL) and4-dimethylamino-pyridine (500 mg) were added. The reaction mixture wasstirred at room temperature for 3 hours and then concentrated underreduced pressure. The residue was dissolved with ethyl acetate (1 L) andsuccessively washed with a saturated aqueous sodium hydrogen carbonatesolution, a 1 M hydrochloric acid and water. The organic layer was driedover sodium sulfate and evaporated under reduced pressure. The resultingresidue was purified by silica gel column chromatography [eluent:stepwise gradient of diethyl ether (30-40%) in petroleum ether] to givepure GG (16.2 g, 60%) as a pale yellow syrup. A small fraction of thematerial was re-purified on silica gel chromatography [same eluent:system] in order separate the α and the β anomers.

α anomer: ¹H-NMR (DMSO-d₆): δ 8.1-7.5 (m, 5H, C₆H₅CO), 6.34 (pt, 1H,H-1, J=2.4 Hz, J=2,1 Hz), 5.49 (m, 2H, H-2 and H-3), 4.33 (q, 2H, H-5and H-5′, J=11.6 Hz, J=18.7 Hz), 2.15 (s, 3H, CH₃CO₂), 2.11 (s, 3H,CH₃CO₂), 2.07 (s, 3H, CH₃CO₂), 1.37 (s, 3H, CH₃); MS (matrix GT): FAB>0m/z 335 (M−CH₃CO₂ ⁻)⁺, 275 (M−CH₃CO₂-+H)⁺, 105 (C₆H₅CO)⁺, 43 (CH₃CO)⁺FAB<0 m/z 121 (C₆H₅CO₂)⁻, 59 (CH₃CO₂)⁻.

β anomer: ¹H-NMR (DMSO-d₆): δ 8.1-7.5 (m, 5H, C₆H₅CO), 5.99 (s, 1H,H-1), 5.46 (d, 1H, H-2, J2-3=5.3 HZ), 5.30 (d, 1H, H-2, J₂₋₃=5.3 Hz),4.39 (d, 1H, H-5, J_(5-5′), =11.7 Hz), 4.19 (d, 1H, H-5′, J_(5′-5)=11.7Hz), 2.10 (s, 3H, CH₃CO₂), 2.06 (s, 3H, CH₃CO₂), 2.02 (s, 3H, CH₃CO₂),1.30 (s, 3H, CH₃); MS (matrix GT): FAB>0 m/z 335 (M−CH₃CO₂ ⁻)⁺, 275(M−CH₃CO₂ ⁻+H)⁺, 105 (C₆H₅CO)⁺, 43 (CH₃CO)+FAB<0 m/z 121 (C₆H₅CO₂)⁻, 59(CH₃CO₂)⁻.

EXAMPLE 11 Preparation of1-(5-O-benzoyl-4-C-methyl-2,3-O-acetyl-O-D-ribofuranosyl)uracil (HH)

A suspension of uracil (422 mg, 3.76 mmol) was treated withhexamethyldisilazane (HMDS, 21 mL) and a catalytic amount of ammoniumsulfate during 17 hours under reflux. After cooling to room temperature,the mixture was evaporated under reduced pressure, and the residue,obtained as a colorless oil, was diluted with anhydrous1,2-dichloroethane (7.5 mL). To the resulting solution was added GG(0.99 g, 2.51 mmol) in anhydrous 1,2-dichloroethane (14 mL), followed byaddition of trimethylsilyl trifluoromethanesulfonate (TMSTf, 0.97 mL,5.02 mmol). The solution was stirred for 2.5 hours at room temperatureunder argon atmosphere, then diluted with chloroform (150 mL), washedwith the same volume of a saturated aqueous sodium hydrogen carbonatesolution and finally with water (2×100 mL). The organic phase was driedover sodium sulfate, then evaporated under reduced pressure. Theresulting crude material was purified by silica gel columnchromatography [eluent: stepwise gradient of methanol (0-2%) inchloroform] to afford pure HH (1.07 g, 95%) as a foam. ¹H-NMR (DMSO-d₆):δ 11.48 (s, 1H, NH), 8.1-7.5 (m, 6H, C₆H₅CO and H-6), 5.94 (d, 1H, H-1′,J_(1′-2′)=3.3 Hz), 5.61 (m, 3H, H-5, H-2′ and H-3′), 4.47 (d, 1H, H-5′,J_(5′-5′)=11.7 Hz), 4.35 (d, 1H, H-5″, J_(5″-5′)=11.7 Hz), 2.12 (s, 3H,CH₃CO₂), 2.09 (s, 3H, CH₃CO₂), 1.38 (s, 3H, CH₃); MS (matrix GT): FAB>0m/z 893 (2M+H)⁺, 447 (M+H)⁺, 335 (S)⁺, 113 (BH₂)⁺, 105 (C₆H₅CO)⁺, 43(CH₃CO)⁺ FAB<0 m/z 891 (2M−H)⁻, 445 (M−H)⁻, 121 (C₆H₅CO₂)⁻, 111 (B)⁻, 59(CH₃CO₂)⁻.

EXAMPLE 12 Preparation of 1-(4-C-methyl-β-D-ribofuranosyl)uracil (II)

The title compound can be prepared according to a published procedurefrom HH (Waga, T.; Nishizaki, T.; et al. Biosci. Biotechnol. Biochem.1993, 57, 1433-1438).

A solution of HH (610 mg, 1.37 mmol) in methanolic ammonia (previouslysaturated at −10° C.) (27 mL) was stirred at room temperature overnight.The solvent was evaporated under reduced pressure and the residue waspartitioned between methylene chloride (40 mL) and water (40 mL). Theaqueous layer was washed with methylene chloride (2×40 mL), concentratedunder reduced pressure and coevaporated several times with absoluteethanol. Recrystallization from a mixture absolute ethanol/methanol gaveII (215 mg, 61%) as a colorless and crystalline solid. Mp: 226-227(dec.) (lit. 227: Ref.6); UV (H₂O): λ_(max)=259 nm (ε=10100),λ_(min)=228 nm (ε=2200); HPLC 99.56%, ¹H-NMR (DMSO-d₆): δ 11.28 (s, 1H,NH), 7.89 (d, 1H, H-6, J₆₋₅=8.1 Hz), 5.80 (d, 1H, H-1′, J_(1′-2′)=7.1Hz), 5.64 (d, 1H, H-5, J₅₋₆=8.1 Hz), 5.24 (d, 1H, OH-2′, J_(OH-2′)=6.5Hz), 5.18 (t, 1H, OH-5′ J_(OH-5′)=J_(OH-5″)=5.2 Hz), 5.01 (d, 1H, OH-3′,J_(OH-3′)=5.0 Hz), 4.28 (dd, 1H, H-2′, J=6.5 Hz, J=12.2 Hz), 3.90 (t,1H, H-3′, J_(3′-2′)=J_(3′-OH′)=5.1 Hz), 3.30 (m, 2H, H-5′ and H-5″),1.06 (s, 3H, CH₃); MS (matrix GT): FAB>0 m/z 517 (2M+H)⁺, 259 (M+H)⁺,147 (S)+FAB<0 m/z 515 (2M−H)⁻, 257 (M−H)⁻.

EXAMPLE 13 Preparation of1-(5-O-benzoyl-4-C-methyl-2,3-O-acetyl-β-D-ribofuranosyl)-4-thio-uracil(JJ)

Lawesson's reagent (926 mg, 2.29 mmol) was added under argon to asolution of HH (1.46 g, 3.27 mmol) in anhydrous 1,2-dichloroethane (65mL) and the reaction mixture was stirred overnight under reflux. Thesolvent was evaporated under reduced pressure and the residue waspurified by silica gel column chromatography [eluent: stepwise gradientof methanol (1-2%) in chloroform] to give pure JJ (1.43 g, 95%) as ayellow foam. ¹H-NMR (DMSO-d₆): δ 12.88 (s, 1H, NH), 8.1-7.5 (m, 6H,C₆H₅CO and H-6), 6.27 (d, 1H, H-1′, J_(1′-2′)=7.51 Hz), 5.91 (br s, 1H,H-5) 5.64 (m, 2H, H-2′ and H-3′), 4.47 (d, 1H, H-5′, J_(5′-5″)=11.7 Hz),4.36 (d, 1H, H-5′, J_(5′-5′)′=11.7 Hz), 2.11 (s, 3H, CH₃CO₂), 2.09 (s,3H, CH₃CO₂), 1.39 (s, 3H, CH₃); MS (matrix GT): FAB>0 m/z 925 (2M+H)⁺,463 (M+H)⁺, 335 (S)⁺, 129 (BH₂)⁺, 105 (C₆H₅CO)⁺, 43 (CH₃CO)⁺ FAB<0 m/z461 (M−H)⁻, 127 (B)⁻, 121 (C₆H₅CO₂)⁻, 59 (CH₃CO₂)⁻.

EXAMPLE 14 Preparation of 1-(4-C-methyl-β-D-ribofuranosyl)4-thio-uracil(KK)

A solution of JJ (500 mg, 1.08 mmol) in methanolic ammonia (previouslysaturated at −10° C.) (27 mL) was stirred at room temperature overnight.The solvent was evaporated under reduced pressure and the residue waspartitioned between methylene chloride (40 ml) and water (40 mL). Theaqueous layer was washed with methylene chloride (2×40 mL), concentratedunder reduced pressure. The crude material was purified by silica gelcolumn chromatography [eluent: stepwise gradient of methanol (5-7%) inmethylene chloride] to give pure KK (188 mg, 63%), which waslyophilized. Mp: 65-70 (dec.); UV (methanol): λ_(max)=330 nm (ε=20000)246 nm (ε=4200), λ_(min)=275 nm (ε=1500); ¹H-NMR (DMSO-d₆): δ 12.51(brs, 1H, NH), 7.81 (d, 1H, H-6, J₆₋₅=7.6 Hz), 6.30 (d, 1H, H-5,J₅₋₆=7.5 Hz), 5.77, (d, 1H, H-1′, J_(1′-2′)=6.7 Hz), 5.32 (d, 1H, OH-2′,J_(OH-2′)=6.1 Hz), 5.20 (t, 1H, OH-5′ J_(OH-5′)=J_(OH-5″)=5.2 Hz), 5.03(d, 1H, OH-3′, J_(OH-3′)=5.2 Hz), 4.17 (dd, 1H, H-2′, J=6.2 Hz, J=12,0Hz), 3.89 (t, 1H, H-3′, J_(3′-2′)=J_(3′-OH′)=5.1 Hz), 3.35 (m, 2H, H-5′and H-5″), 1.02 (s, 3H, CH₃); MS (matrix GT): FAB>0 m/z 275 (M+H)⁺, 147(S)⁺, 129(BH₂)⁺ FAB<0 m/z 547 (2M−H)⁻, 273 (M−H)⁻, 127 (B)⁻.

EXAMPLE 15 Preparation of 1-(4-C-methyl-β-D-ribofuranosyl)cytosinehydrochloric form (LL)

Compound KK (890 mg, 1.93 mmol) was treated with methanolic ammonia(previously saturated at −10° C.), (12 mL) at 100° C. in astainless-steel bomb for 3 hours, then cooled to room temperature. Thesolvent was evaporated under reduced pressure and the residue waspartitioned between methylene chloride (40 mL) and water (40 mL). Theaqueous layer was washed with methylene chloride (2×40 mL), concentratedunder reduced pressure. The crude material was purified by silica gelcolumn chromatography [eluent: methylene chloride/methanol/ammoniumhydroxide 65:30:5]. The collected fractions were evaporated underreduced pressure and in absolute ethanol (6.3 mL). To the solution wasadded a 2N hydrochloric acid solution (1.5 mL) and the mixture wasstirred before being concentrated under reduced pressure. The procedurewas repeated twice and LL was precipitated from absolute ethanol. Mp:213-214 (dec.); UV (methanol): λ_(max)=280 nm (ε=9800), λ_(min)=245 nm(ε=3600); ¹H-NMR (DMSO-d₆): δ 9.82 (s, 1H, NH₂), 8.72 (s, 1H, NH₂), 8.34(d, 1H, H-6, J₆₋₅=7.8 Hz), 6.21 (d, 1H, H-5, J₅₋₆=7.8 Hz), 5.83 (d, 1H,H-1′, J_(1′-2′)=5.8 Hz), 4.22 (d, 1H, OH-2′, J_(OH-2′)=6.5 Hz), 5.6-4.7(m, 3H, OH-2′, OH-3′ and OH-5′), 4.28 (t, 1H, H-2′, J=5.6 Hz), 3.99 (d,1H, H-3′, J=5.3 Hz), 3.43 (m, 2H, H-5′ and H-5″), 1.14 (s, 3H, CH₃); MS(matrix GT): FAB>0 m/z 515 (2M+H)⁺, 258 (M+H)⁺, 147 (S)⁺, 112 (BH₂)⁺FAB<0 m/z 256 (M−H)⁻.

EXAMPLE 16 Preparation of1-(5-O-benzoyl-4-C-methyl-2,3-O-acetyl-β-D-ribofuranosyl)thymine (MM)

A suspension of thymine (384 mg, 3.04 mmol) was treated withhexamethyldisilazane (HMDS, 17 mL) and a catalytic amount of ammoniumsulfate overnight under reflux. After cooling to room temperature, themixture was evaporated under reduced pressure, and the residue, obtainedas a colorless oil, was diluted with anhydrous 1,2-dichloroethane (6mL). To the resulting solution was added GG (1.0 g, 2.53 mmol) inanhydrous 1,2-dichloroethane (14 mL), followed by addition oftrimethylsilyl trifluoromethanesulfonate (TMSTf, 0.98 mL, 5.06 mmol).The solution was stirred for 5 hours at room temperature under argonatmosphere, then diluted with chloroform (150 mL), washed with the samevolume of a saturated aqueous sodium hydrogen carbonate solution andfinally with water (2×100 mL). The organic phase was dried over sodiumsulfate, then evaporated under reduced pressure. The resulting crudematerial was purified by silica gel column chromatography [eluent: 2% ofmethanol in chloroform] to afford pure MM (1.09 g, 94%) as a foam.¹H-NMR (DMSO-d₆): δ 11.47 (s, 1H, NH), 8.1-7.4 (m, 6H, C₆H₅CO and H-6),5.98 (d, 1H, H-1′, J=5.0 Hz), 5.5-5.7 (m, 2H, H-2′ and H-3′), 4.42 (dd,2H, H-5′ and H-5″, J=11.6 Hz, J=31.6 Hz), 2.12 (s, 3H, CH₃CO₂), 2.09 (s,3H, CH₃CO₂), 1.60 (s, 1H, CH₃), 1.37 (s, 3H, CH₃); MS (matrix GT): FAB>0m/z 461 (M+H)⁺, 335 (S)⁺, 105 (C₆H₅CO)⁺, 43 (CH₃CO)+FAB<0 m/z 459(M−H)⁻, 125 (B)⁻, 121 (C₆H₅CO₂)⁻, 59 (CH₃CO₂)⁻.

EXAMPLE 17 Preparation of 1-(4-C-methyl-β-D-ribofuranosyl)thymine (NN)

The title compound can be prepared according to a published procedurefrom MM (Waga, T.; Nishizaki, T.; et al. Biosci. Biotechnol. Biochem.1993, 57, 1433-1438).

A solution of MM (1.09 g, 2.37 mmol) in methanolic ammonia (previouslysaturated at −10° C.) (60 mL) was stirred at room temperature overnight.The solvent was evaporated under reduced pressure and the residue waspartitioned between methylene chloride (60 mL) and water (60 mL). Theaqueous layer was washed with methylene chloride (2×60 mL), concentratedunder reduced pressure and coevaporated several times with absoluteethanol. Recrystallization from methanol gave NN (450 mg, 70%) as acolorless and crystalline solid. Mp: 258-260 (dec.) (lit. 264: Ref.6);UV (H₂O): λ_(max)=264.4 nm (ε=8800), λ_(min)=232.0 nm (ε=2200); ¹H-NMR(DMSO-d₆): δ 11.29 (s, 1H, NH), 7.75 (s, 1H, H-6), 5.82 (d, 1H, H-1′,J_(1′-2′)=7.2 Hz), 5.19 (m, 2H, OH-2′, OH-5′), 5.02 (d, 1H, OH-3′,J_(OH-3′)=5.0 Hz), 4.21 (dd, 1H, H-2′, J=6.4 Hz, J=12.3 Hz), 3.92 (t,1H, H-3′, J_(3′-2′)=J_(3′-OH′)=5.0 Hz), 3.30 (m, 2H, H-5′ and H-5″),1.78 (s, 3H, CH₃), 1.09 (s, 3H, CH₃); MS (matrix GT): FAB>0 m/z 545(2M+H)⁺, 365 (M+G+H)⁺, 273 (M+H)⁺, 147 (S)⁺, 127 (B+2H)⁺, FAB<0 m/z 543(2M−H)⁻, 271 (M−H)⁻, 125 (B)⁻; [α]_(D) ²⁰−32.0 (c=0.5 in H₂O, litt.−26.4).

EXAMPLE 18 Preparation of1-(5,2,3-tri-O-acetyl-4-C-methyl-β-D-ribofuranosyl)thymine (O)

A solution of NN (200 mg, 0.735 mmol) in anhydrous pyridine (7.4 ml) wastreated with acetic anhydride (1.2 mL) and stirred at room temperaturefor 3 hours. The solvent was evaporated under reduced pressure, and theresidue was purified by silica gel column chromatography [eluent:stepwise gradient of methanol (0-5%) in methylene chloride] to affordpure OO (0.400 g, quantitative yield) as a foam. ¹H-NMR (DMSO-d₆): δ11.45 (s, 1H, NH), 7.56 (s, 1H, H-6), 5.90 (d, 1H, H-1′, J_(1′-2′)=4.8Hz), 5.5-5.4 (m, 2H, H-2′ and H-3′), 4.3-4.0 (m, 2H, H-5′ and H-5″),2.1-2.0 (m, 9H, 3 CH₃CO₂), 1.78 (s, 1H, CH₃), 1.20 (s, 3H, CH₃); MS(matrix GT): FAB>0 m/z 797 (2M+H)⁺, 399 (M+H)⁺, 339 (M−CH₃CO₂)⁺, 273(S)⁺, 127 (BH₂)⁺, 43 (CH₃CO)⁺ FAB<0 m/z 795 (2M−H)⁻, 397 (M−H)⁻, 355(M−CH₃CO)⁻, 125 (B)⁻, 59 (CH₃CO₂)⁻.

EXAMPLE 19 Preparation of1-(5,2,3-tri-O-acetyl-4-C-methyl-β-D-ribofuranosyl)-4-thio-thymine (PP)

Lawesson's reagent (119 mg, 0.29 mmol) was added under argon to asolution of OO (0.167 g, 4.19 mmol) in anhydrous 1,2-dichloroethane (11mL) and the reaction mixture was stirred overnight under reflux. Thesolvent was evaporated under reduced pressure and the residue waspurified by silica gel column chromatography [eluent: stepwise gradientof methanol (1-2%) in chloroform] to give pure PP (0.165 g, 95%) as ayellow foam. ¹H-NMR (DMSO-d₆): δ 12.81 (s, 1H, NH), 7.64 (s, 1H, H-6),5.84(d, 1H, H-1′, J_(1′-2′)=4.66 Hz), 5.5-5.4 (m, 2H, H-2′ and H-3′),4.11 (dd, 2H, H-5′ and H-5″, J=11.7 Hz, J=31.3 Hz), 2.0-1.8 (m, 12H, 3CH₃CO₂ and CH₃), 1.33 (s, 3H, CH₃); MS (matrix GT): FAB>0 m/z 829(2M+H)⁺, 415 (M+H)⁺, 273 (S)⁺, 143 (BH₂)⁺, 43 (CH₃CO)+FAB<0 m/z 827(2M−H)⁻, 413 (M−H)⁻, 141 (B)⁻, 59 (CH₃CO₂)⁻.

In a similar manner, the following nucleosides of Formula XVII areprepared, using the appropriate sugar and pyrimidine bases.

wherein R¹, R², R³, X¹ and Y are defined in Table 19.

EXAMPLE 20 Preparation of1-(4-C-methyl-β-D-ribofuranosyl)-5-methyl-cytosine (QQ), hydrochlorideform

Compound PP (0.160 g, 0.386 mmol) was treated with methanolic ammonia(previously saturated at −10° C.), (10 mL) at 100° C. in astainless-steel bomb for 3 hours, then cooled to room temperature. Thesolvent was evaporated under reduced pressure and the residue waspartitioned between methylene chloride (30 mL) and water (30 mL). Theaqueous layer was washed with methylene chloride (2×30 mL), concentratedunder reduced pressure. The crude material was purified by silica gelcolumn chromatography [eluent: 20% methanol in methylene chloride] toafford 1-(4-C-methyl-β-D-ribofuranosyl)-5-methyl-cytosine (60 mg, 57%).This compound was dissolved in EtOH 100 (1.5 mL), treated with a 2Nhydrochloric acid solution (0.3 mL), and the mixture was stirred beforebeing concentrated under reduced pressure. The procedure was repeatedtwice and QQ was precipitated from absolute ethanol. Mp: 194-200 (dec.);UV (H₂O): λ_(max)=275.6 nm (ε=7300), λ_(min)=255 nm (ε=4700); HPLC 100%,¹H-NMR (DMSO-d₆): δ 9.34 and 9.10 (2s, 2H, NH₂), 8.21 (s, 1H, H-6), 5.80(d, 1H, H-1′, J_(1′-2′)=6.0 Hz), 5.3-4.3 (m, 3H, OH-2′, OH-3′ andOH-5′), 4.21 (t, 1H, H-2′, J=5.7 Hz), 3.98 (d, 1H, H-3′, J=5.3 Hz),3.5-3.3 (m, 2H, H-5′ and H-5″), 1.97 (s, 3H, CH₃), 1.12 (s, 3H, CH₃).

EXAMPLE 21 Preparation OFO-6-diphenylcarbamoyl-N²-isobutyryl-9-(2,3-di-O-acetyl-5-O-benzoyl-4-C-methyl-β-D-ribofuranosyl)guanine(RR)

To a suspension of O-6-diphenylcarbamoyl-N²-isobutyrylguanine (1.80 g,4.33 mmol) in anhydrous toluene (20 mL) was addedN,O-bis(trimethylsilyl)acetamide (1.92 mL, 7.9 mmol). The reactionmixture was allowed to warm under reflux for 1 hour. Compound GG (1.55g, 3.93 mmol) was dissolved in toluene (10 mL) andtrimethylsilyltrifluoromethanesulfonate (TMSTf) (915 mL, 4.72 mmol) wasadded. The mixture was heated under reflux for 30 minutes. The solutionwas then cooled to room temperature and neutralized with a 5% aqueoussodium hydrogen carbonate solution. The reaction mixture was dilutedwith ethyl acetate (200 mL). The organic phase was washed with a 5%aqueous sodium hydrogen carbonate solution (150 mL) and with water(2×150 mL). The organic layer was dried over Na₂SO₄ and evaporated todryness. The residue was purified by silica gel column chromatography[eluent: stepwise gradient of diethyl ether (70-90%) in petroleum ether]to afford pure RR (1.62 g, 55%) as a foam.

EXAMPLE 22 Preparation of 9-(4-C-methyl-β-D-ribofuranosyl)guanine (SS)

The title compound can be prepared according to a published procedurefrom RR (Waga, T.; Nishizaki, T.; et al. Biosci. Biotechnol. Biochem.1993, 57, 1433-1438).

A solution of RR (1.50 g, mmol) in methanolic ammonia (previouslysaturated at −10° C.) (20 mL) was stirred at room temperature overnight.The solvent was evaporated under reduced pressure and the residue waspartitioned between methylene chloride (60 mL) and water (60 mL). Theaqueous layer was washed with methylene chloride (2×60 mL), concentratedunder reduced pressure. The residue was purified by an RP18 columnchromatography [eluent water/acetonitrile 95/5] to afford pure SS (380mg, 60%). Recrystallization from water gave S as a crystalline solid.Mp>300 (dec.), UV (H₂O): λ_(max)=252 nm (ε=14500), ¹H-NMR (DMSO-d₆): δ10.64 (s, 1H, NH), 7.95 (s, 1H, H-8), 6.45 (s1, 2H, NH₂), 5.68 (d, 1H,H-1′, J_(1′-2′)=7.45 Hz), 5.31 (d, 1H, OH, OH-2′, J_(OH-2′)=6.8 Hz),5.17 (t, 1H, OH, OH-5′, J=5.5 Hz), 5.07 (d, 1H, OH-3′, J_(OH-3′)=4.5Hz), 4.65 (dd, 1H, H-2′, J=7.1 Hz, J=12.2 Hz), 4.00 (t, 1H, H-3′,J_(3′-2′)=J_(3′-OH′)=4.8 Hz), 3.41 (m, 2H, H-5′ and H-5″), 1.12 (s, 3H,CH₃); MS (matrix GT): FAB>0 m/z 595 (2M+H)⁺, 390 (M+G+H)⁺, 298 (M+H)⁺,152 (B+2H)⁺, FAB<0 m/z 593 (2M−H)⁻, 296 (M−H)⁻, 150 (B)⁻.

EXAMPLE 239-(2,3-di-O-acetyl-5-O-benzoyl-4-C-methyl-β-D-ribofuranosyl)adenine (TT)

A solution of GG (1.10 g, 2.79 mmol) in anhydrous acetonitrile (50 ml)was treated with adenine (452.4 mg, 3.35 mmol) and stannic chloride(SnCl₄, 660 μL, 5.58 mmol) and stirred at room temperature overnight.The solution was concentrated under reduced pressure, diluted withchloroform (100 mL) and treated with a cold saturated aqueous solutionof NaHCO₃ (100 ml). The mixture was filtered on celite, and theprecipitate was washed with hot chloroform. The filtrates were combined,washed with water (100 ml) and brine (100 ml), dried (Na₂SO₄), andevaporated under reduced pressure. The residue was purified by silicagel column chromatography [eluent: stepwise gradient of methanol (3-5%)in dichloromethane] to afford pure TT (977 mg, 77%) as a white foam.¹H-NMR (DMSO-d₆): δ 8.31-7.49 (m, 7H, C₆H₅CO, H-2 and H-8), 7.37 (1s,2H, NH₂) 6.27 (m, 2H, H-1′ and H-3′), 5.90 (m, 1H, H-2′), 4.60 (d, 1H,H-5′, J=11.7 Hz), 4.35 (d, 1H, H-5″), 2.17 (s, 3H, CH₃CO₂), 2.06 (s, 3H,CH₃CO₂), 1.42 (s, 3H, CH₃).

EXAMPLE 24 Preparation of 9-(4-C-methyl-β-D-ribofuranosyl)adenine (UU)

The title compound can be prepared according to a published procedurefrom TT (Waga, T.; Nishizaki, T.; et al. Biosci. Biotechnol. Biochem.1993, 57, 1433-1438).

A solution of TT (970 mg, 2.08 mmol) in methanolic ammonia (previouslysaturated at −10° C.) (50 mL) was stirred at room temperature overnight.The solvent was evaporated under reduced pressure and the residue waspartitioned between methylene chloride (100 ml) and water (100 ml). Theaqueous layer was washed with methylene chloride (2×100 mL), andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography [eluent: stepwise gradient of methanol(10-30%) in ethyl acetate] to afford pure U (554 mg, 95%).Crystallization from methanol/ethyl acetate gave UU as a white solid.Mp: 96-97 (dec.); ¹H-NMR (DMSO-d₆): δ 8.33 (s, 1H, H-2), 8.13 (s, 1H,H-8), 7.36 (brs, 2H, NH2), 5.84 (d, 1H, H-1′, J_(1′-2′)=7.4 Hz), 5.69(dd, 1H, OH-5′, J=4.2 Hz and J=7.8 Hz), 5.33 (d, 1H, OH-2′, J=6.6 Hz),5.13 (d, 1H, OH-3′, J=4.4 Hz), 4.86 (m, 1H, H-2′), 4.04 (t, 1H, H-3′),3.58-3.32 (m, 2H, H-5′ and H-5″), 1.15 (s, 3H, CH₃); MS (matrix GT):FAB>0 m/z 563 (2M+H)⁺, 374 (M+G+H)⁺, 282 (M+H)⁺, 136 (B+2H)⁺, FAB<0 m/z561 (2M−H)⁻, 280 (M−H)⁻, 134 (B)⁻.

In a similar manner, the following nucleosides of Formula XVI areprepared, using the appropriate sugar and purine bases.

wherein R¹, R², R³, X¹, X², and Y are defined in Table 20.

Alternatively, the following nucleosides of Formula XVIII are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R³, R⁶, X and Base are defined in Table 21.

Alternatively, the following nucleosides of Formula XIX are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R⁶, X and Base are defined in Table 22.

Alternatively, the following nucleosides of Formula XXIV are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R², R⁶, X, and Base are defined in Table 23.

Alternatively, the following nucleosides of Formula XX are prepared,using the appropriate sugar and pyrimidine or purine bases.

wherein R¹, R⁶, R⁷, R⁸, X, Base, R¹⁰ and R⁹ are defined in Table 24.

Tables 1-24 set out examples of species within the present invention.When the amino acid appears in the table, it is considered to be aspecific and independent disclosure of each of the esters of α, β γ or δglycine, alanine, valine, leucine, isoleucine, methionine,phenylalanine, tryptophan, proline, serine, threonine, cysteine,tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginineand histidine in the D and L-configurations. When the term acyl is usedin the tables, it is meant to be a specific and independent disclosureof any of the acyl groups as defined herein, including but not limitedto acetyl, trifluoroacetyl, methylacetyl, cyclopropylacetyl,cyclopropylcarboxy, propionyl, butyryl, hexanoyl, heptanoyl, octanoyl,neo-heptanoyl, phenylacetyl, diphenylacetyl,α-trifluoromethyl-phenylacetyl, bromoacetyl, 4-chloro-benzeneacetyl,2-chloro-2,2-diphenylacetyl, 2-chloro-2-phenylacetyl, trimethylacetyl,chlorodifluoroacetyl, perfluoroacetyl, fluoroacetyl,bromodifluoroacetyl, 2-thiopheneacetyl, tert-butylacetyl,trichloroacetyl, monochloro-acetyl, dichloroacetyl, methoxybenzoyl,2-bromo-propionyl, decanoyl, n-pentadecanoyl, stearyl,3-cyclopentyl-propionyl, 1-benzene-carboxyl, pivaloyl acetyl,1-adamantane-carboxyl, cyclohexane-carboxyl, 2,6-pyridinedicarboxyl,cyclopropane-carboxyl, cyclobutane-carboxyl, 4-methylbenzoyl, crotonyl,1-methyl-1H-indazole-3-carbonyl, 2-propenyl, isovaleryl,4-phenylbenzoyl.

F. Biological Assays

Compounds can exhibit anti-flavivirus or pestivirus activity byinhibiting flavivirus or pestivirus polymerase, by inhibiting otherenzymes needed in the replication cycle, or by other pathways.

Phosphorylation Assay of Nucleoside to Active Triphosphate

To determine the cellular metabolism of the compounds, HepG2 cells wereobtained from the American Type Culture Collection (Rockville, Md.), andwere grown in 225 cm² tissue culture flasks in minimal essential mediumsupplemented with non-essential amino acids, 1% penicillin-streptomycin.The medium was renewed every three days, and the cells were subculturedonce a week. After detachment of the adherent monolayer with a 10 minuteexposure to 30 mL of trypsin-EDTA and three consecutive washes withmedium, confluent HepG2 cells were seeded at a density of 2.5×10⁶ cellsper well in a 6-well plate and exposed to 10 μM of [³H] labeled activecompound (500 dpm/pmol) for the specified time periods. The cells weremaintained at 37° C. under a 5% CO₂ atmosphere. At the selected timepoints, the cells were washed three times with ice-coldphosphate-buffered saline (PBS). Intracellular active compound and itsrespective metabolites were extracted by incubating the cell pelletovernight at −20° C. with 60% methanol followed by extraction with anadditional 20 μL of cold methanol for one hour in an ice bath. Theextracts were then combined, dried under gentle filtered air flow andstored at −20° C. until HPLC analysis.

Bioavailability Assay in Cynomolgus Monkeys

Within 1 week prior to the study initiation, the cynomolgus monkey wassurgically implanted with a chronic venous catheter and subcutaneousvenous access port (VAP) to facilitate blood collection and underwent aphysical examination including hematology and serum chemistryevaluations and the body weight was recorded. Each monkey (six total)receives approximately 250 μCi of ³H activity with each dose of activecompound at a dose level of 10 mg/kg at a dose concentration of 5 mg/mL,either via an intravenous bolus (3 monkeys, IV), or via oral gavage (3monkeys, PO). Each dosing syringe was weighed before dosing togravimetrically determine the quantity of formulation administered.Urine samples were collected via pan catch at the designated intervals(approximately 18-0 hours pre-dose, 0-4, 4-8 and 8-12 hours post-dosage)and processed. Blood samples were collected as well (pre-dose, 0.25,0.5, 1, 2, 3, 6, 8, 12 and 24 hours post-dosage) via the chronic venouscatheter and VAP or from a peripheral vessel if the chronic venouscatheter procedure should not be possible. The blood and urine sampleswere analyzed for the maximum concentration (C_(max)), time when themaximum concentration was achieved (T_(max)), area under the curve(AUC), half life of the dosage concentration (T_(1/2)), clearance (CL),steady state volume and distribution (V_(ss)) and bioavailability (F).

Bone Marrow Toxicity Assay

Human bone marrow cells were collected from normal healthy volunteersand the mononuclear population were separated by Ficoll-Hypaque gradientcentrifugation as described previously by Sommadossi J-P, Carlisle R.Antimicrobial Agents and Chemotherapy 1987; 31:452-454; and SommadossiJ-P, Schinazi R F, et al. Biochemical Pharmacology 1992; 44:1921-1925.The culture assays for CFU-GM and BFU-E were performed using a bilayersoft agar or methylcellulose method. Drugs were diluted in tissueculture medium and filtered. After 14 to 18 days at 37° C. in ahumidified atmosphere of 5% CO₂ in air, colonies of greater than 50cells were counted using an inverted microscope. The results arepresented as the percent inhibition of colony formation in the presenceof drug compared to solvent control cultures.

Mitochondria Toxicity Assay

HepG2 cells were cultured in 12-well plates as described above andexposed to various concentrations of drugs as taught by Pan-Zhou X-R,Cui L, et al. Antimicrob. Agents Chemother. 2000; 44:496-503. Lacticacid levels in the culture medium after 4 day drug exposure weremeasured using a Boehringer lactic acid assay kit. Lactic acid levelswere normalized by cell number as measured by hemocytometer count.

Cytotoxicity Assay

Cells were seeded at a rate of between 5×10³ and 5×10⁴/well into 96-wellplates in growth medium overnight at 37° C. in a humidified CO₂ (5%)atmosphere. New growth medium containing serial dilutions of the drugswas then added. After incubation for 4 days, cultures were fixed in 50%TCA and stained with sulforhodamineB. The optical density was read at550 nm. The cytotoxic concentration was expressed as the concentrationrequired to reduce the cell number by 50% (CC₅₀).

Cell Protection Assay (CPA)

The assay was performed essentially as described by Baginski, S. G.;Pevear, D. PNAS USA 2000, 97(14), 7981-7986. MDBK cells (ATCC) wereseeded onto 96-well culture plates (4,000 cells per well) 24 hoursbefore use. After infection with BVDV (strain NADL, ATCC) at amultiplicity of infection (MOI) of 0.02 plaque forming units (PFU) percell, serial dilutions of test compounds were added to both infected anduninfected cells in a final concentration of 0.5% DMSO in growth medium.Each dilution was tested in quadruplicate. Cell densities and virusinocula were adjusted to ensure continuous cell growth throughout theexperiment and to achieve more than 90% virus-induced cell destructionin the untreated controls after four days post-infection. After fourdays, plates were fixed with 50% TCA and stained with sulforhodamine B.The optical density of the wells was read in a microplate reader at 550nm. The 50% effective concentration (EC₅₀) values were defined as thecompound concentration that achieved 50% reduction of cytopathic effectof the virus.

Plaque Reduction Assay

For each compound the effective concentration was determined induplicate 24-well plates by plaque reduction assays. Cell monolayerswere infected with 100 PFU/well of virus. Then, serial dilutions of testcompounds in MEM supplemented with 2% inactivated serum and 0.75% ofmethyl cellulose were added to the monolayers. Cultures were furtherincubated at 37° C. for 3 days, then fixed with 50% ethanol and 0.8%Crystal Violet, washed and air-dried. Then plaques were counted todetermine the concentration to obtain 90% virus suppression.

Yield Reduction Assay

For each compound the concentration to obtain a 6-log reduction in viralload was determined in duplicate 24-well plates by yield reductionassays. The assay was performed as described by Baginski, S. G.; Pevear,D. PNAS USA 2000, 97(14), 7981-7986, with minor modifications. Briefly,MDBK cells were seeded onto 24-well plates (2×105 cells per well) 24hours before infection with BVDV (NADL strain) at a multiplicity ofinfection (MOI) of 0.1 PFU per cell. Serial dilutions of test compoundswere added to cells in a final concentration of 0.5% DMSO in growthmedium. Each dilution was tested in triplicate. After three days, cellcultures (cell monolayers and supernatants) were lysed by threefreeze-thaw cycles, and virus yield was quantified by plaque assay.Briefly, MDBK cells were seeded onto 6-well plates (5×105 cells perwell) 24 h before use. Cells were inoculated with 0.2 mL of test lysatesfor 1 hour, washed and overlaid with 0.5% agarose in growth medium.After 3 days, cell monolayers were fixed with 3.5% formaldehyde andstained with 1% crystal violet (w/v in 50% ethanol) to visualizeplaques. The plaques were counted to determine the concentration toobtain a 6-log reduction in viral load.

EXAMPLE 25 Antiviral Potency of Test Compounds in a Cell Based Assay

The titer of BVDB (Log₁₀ units/ml) were identified after treatment ofinfected MDBK cells with increasing concentrations of four testcompounds. Ribavirin was used as a standard. This data is shown in FIG.11. The graph shows the antiviral potency of these compounds.

EXAMPLE 26 Cellular Pharmacology of 2′-C-methyl-cytidine-3′-O-L-valineester (Val-mCyd)

Phosphorylation Assay of Nucleoside to Active Triphosphate

To determine the cellular metabolism of the compounds, HepG2 cells wereobtained from the American Type Culture Collection (Rockville, Md.), andwere grown in 225 cm² tissue culture flasks in minimal essential mediumsupplemented with non-essential amino acids, 1% penicillin-streptomycin.The medium was renewed every three days, and the cells were subculturedonce a week. After detachment of the adherent monolayer with a 10 minuteexposure to 30 mL of trypsin-EDTA and three consecutive washes withmedium, confluent HepG2 cells were seeded at a density of 2.5×10⁶ cellsper well in a 6-well plate and exposed to 10 μM of [³H] labeled activecompound (500 dpm/pmol) for the specified time periods. The cells weremaintained at 37° C. under a 5% CO₂ atmosphere. At the selected timepoints, the cells were washed three times with ice-coldphosphate-buffered saline (PBS). Intracellular active compound and itsrespective metabolites were extracted by incubating the cell pelletovernight at −20° C. with 60% methanol followed by extraction with anadditional 20 μL of cold methanol for one hour in an ice bath. Theextracts were then combined, dried under gentle filtered air flow andstored at −20° C. until HPLC analysis.

Antiviral nucleosides and nucleoside analogs were generally convertedinto the active metabolite, the 5′-triphosphate (TP) derivatives byintracellular kinases. The nucleoside-TPs then exert their antiviraleffect by inhibiting the viral polymerase during virus replication. Inprimary human hepatocyte cultures, in a human hepatoma cell line(HepG2), and in a bovine kidney cell line (MDBK), mCyd was convertedinto a major metabolite, 2′-C-methyl-cytidine-5′-triphosphate (mCyd-TP),along with smaller amounts of a uridine 5′-triphosphate derivative,2′-C-methyl-uridine-5′-triphosphate (mUrd-TP). mCyd-TP is inhibitorywhen tested in vitro against the BVDV replication enzyme, the NS5B RNAdependent RNA polymerase, and is thought to be responsible for theantiviral activity of mCyd.

The cellular metabolism of mCyd was examined using MDBK cells, HepG2cells and human primary hepatocytes exposed to 10 μM [³H]-mCyd.High-pressure liquid chromatography (HPLC) analysis demonstrated thatmCyd was phosphorylated in all three cell types, with mCyd-TP being thepredominant metabolite after 24 h. The metabolic profile obtained over a48-hour exposure of human hepatoma HepG2 cells to 10 μM [³H]-mCyd wastested. In HepG2 cells, levels of mCyd-TP peaked at 41.5±13.4 μM after24 hours (see Table 25) and fell slowly thereafter. In primary humanhepatocytes, the peak mCyd-TP concentration at 24 hours was 4 fold lowerat 10.7±6.7 μM. MDBK bovine kidney cells yielded intermediate levels ofmCyd-TP (30.1±6.9 μM at 24 hours).

Exposure of hepatocytes to mCyd led to production of a second5′-triphosphate derivative, mUrd-TP. In HepG2 cells exposed to 10 μM[³H]-mCyd, the mUrd-TP level reached 1.9±1.6 μM at 24 hours, compared to8.1±3.4 μM in MDBK cells and 3.2±2.0 μM in primary human hepatocytes.While MDBK and HepG2 cells produced comparable total amounts ofphosphorylated species (approximately 43 versus 47 μM, respectively) at24 h, mUrd-TP comprised 19% of the total product in MDBK cells versusonly 4% in HepG2 cells. mUrd-TP concentration increased steadily overtime, however reached a plateau or declined after 24 hours. TABLE 25Activation of mCyd (10 μM) in Hepatocytes and MDBK Cells Metabolite (μM)Cells^(a) n mCyd-MP mUrd-MP mCyd-DP mUrd-DP mCyd-TP mUrd-TP HepG2 6 NDND  3.7 ± 2.1 ND  41.5 ± 13.4 1.9 ± 1.6 Human 5 ND ND 1.15 ± 1.1 0.26 ±0.4  10.7 ± 6.7 3.2 ± 2.0 Primary C Hepatocytes MDBK 7 ND ND 4.2 ± 2.70.76 ± 0.95 30.1 ± 6.9 8.1 ± 3.4 Bovine Kidney Cells^(a)Cells were incubated for 24 hours with [³H]-mCyd, specific activity:HepG2 assay = 0.5 Ci/mmol; human and monkey hepatocyte assay = 1.0Ci/mmol.b. The concentrations of metabolites were determined as pmoles permillion cells. One pmole per million cells is roughly equivalent to 1μM.ND, not detected.

The apparent intracellular half-life of the mCyd-TP was 13.9±2.2 hoursin HepG2 cells and 7.6±0.6 hours in MDBK cells: the data were notsuitable for calculating the half life of mUrd-TP. Other than thespecific differences noted above, the phosphorylation pattern detectedin primary human hepatocytes was qualitatively similar to that obtainedusing HepG2 or MDBK cells.

EXAMPLE 27 Cell Cytotoxicity

Mitochondria Toxicity Assay

HepG2 cells were cultured in 12-well plates as described above andexposed to various concentrations of drugs as taught by Pan-Zhou X-R,Cui L, et al. Antimicrob. Agents Chemother. 2000; 44:496-503. Lacticacid levels in the culture medium after 4 day drug exposure weremeasured using a Boehringer lactic acid assay kit. Lactic acid levelswere normalized by cell number as measured by hemocytometer count.

Cytotoxicity Assays

Cells were seeded at a rate of between 5×10³ and 5×10⁴/well into 96-wellplates in growth medium overnight at 37° C. in a humidified CO₂ (5%)atmosphere. New growth medium containing serial dilutions of the drugswas then added. After incubation for 4 days, cultures were fixed in 50%TCA and stained with sulforhodamineB. The optical density was read at550 nm. The cytotoxic concentration was expressed as the concentrationrequired to reduce the cell number by 50% (CC₅₀).

Conventional cell proliferation assays were used to assess thecytotoxicity of mCyd and its cellular metabolites in rapidly dividingcells. The inhibitory effect of mCyd was determined to be cytostatic innature since mCyd showed no toxicity in confluent cells atconcentrations far in excess of the corresponding CC₅₀ for a specificcell line. mCyd was not cytotoxic to rapidly growing Huh7 human hepatomacells or H9c2 rat myocardial cells at the highest concentration tested(CC₅₀>250 μM). The mCyd CC₅₀ values were 132 and 161 μM in BHK-21hamster kidney and HepG2 human hepatoma cell lines, respectively. TheCC₅₀ for mCyd in HepG2 cells increased to 200 μM when the cells weregrown on collagen-coated plates for 4 or 10 days. For comparison, CC₅₀values of 35-36 μM were derived when ribavirin was tested in HepG2 andHuh7 cells. In the MDBK bovine kidney cells used for BVDV antiviralstudies, the CC₅₀ of mCyd was 36 μM. A similar CC₅₀ value (34 μM) wasdetermined for mCyd against MT-4 human T-lymphocyte cells. In addition,mCyd was mostly either non-cytotoxic or weakly cytotoxic (CC₅₀>50to >200 μM) to numerous other cell lines of human and other mammalianorigin, including several human carcinoma cell lines, in testingconducted by the National Institutes of Health (NIH) Antiviral Researchand Antimicrobial Chemistry Program. Exceptions to this were rapidlyproliferating HFF human foreskin fibroblasts and MEF mouse embryofibroblasts, where mCyd showed greater cytotoxicity (CC₅₀s 16.9 and 2.4μM, respectively). Again, mCyd was much less toxic to stationary phasefibroblasts.

The cytotoxic effect of increasing amounts of mCyd on cellular DNA orRNA synthesis was examined in HepG2 cells exposed to [³H]-thymidine or[³H]-uridine. In HepG2 cells, the CC₅₀s of mCyd required to cause 50%reductions in the incorporation of radiolabeled thymidine and uridineinto cellular DNA and RNA, were 112 and 186 μM, respectively. The CC₅₀values determined for ribavirin (RBV) for DNA and RNA synthesis,respectively, were 3.16 and 6.85 μM. These values generally reflect theCC₅₀s of 161 and 36 μM determined for mCyd and RBV, respectively, inconventional cell proliferation cytotoxicity assays. To assess theincorporation of mCyd into cellular RNA and DNA, HepG2 cells wereexposed to 10 μM [³H]-mCyd or control nucleosides (specific activity5.6-8.0 Ci/mmole, labeled in the base) for 30 hours. Labeled cellularRNA or DNA species were separately isolated and incorporation wasdetermined by scintillation counting. Exposure of HepG2 cells to mCydresulted in very low levels of incorporation of the ribonucleosideanalog into either cellular DNA or RNA (0.0013-0.0014 pmole/μg ofnucleic acid). These levels were similar to the 0.0009 and 0.0013pmole/μg values determined for the incorporation of ZDV and ddC,respectively, into RNA: since these deoxynucleosides were not expectedto incorporate into RNA, these levels likely reflect the assaybackground. The incorporation of ZDV and ddC into DNA was significantlyhigher (0.103 and 0.0055 pmole/μg, respectively). Ribavirin (RBV)incorporated into both DNA and RNA at levels 10-fold higher than mCyd.TABLE 26a Cellular Nucleic Acid Synthesis and Incorporation Studies inHepG2 Cells (10 μM Drug and Nucleoside Controls) CC₅₀ (μM) DNA RNAIncorporated drug amount Compound ([³H]Thymidine) ([³H]Uridine) pmole/μgDNA pmole/μg RNA mCyd 112.3 ± 34.5 186.1 ± 28.2 0.0013 ± 0.0008^(a)0.0014 ± 0.0008^(a) ZDV nd nd  0.103 ± 0.0123^(a) 0.0009 ± 0.0003^(a)ddC nd nd 0.0055^(b) 0.0013^(b) Ribavirin  3.16 ± 0.13  6.85 ± 1.830.0120^(b) 0.0132^(c)^(a)Data represent mean of three experiments^(b)Data represent one experiment^(c)Data represent mean of two experimentsnd, not determined

TABLE 26b Cytotoxicity of mCyd in Mammalian Cell Lines Cell Line^(a) nCC₅₀ (μM) Huh 7 7 >250 Hep G2 6 161 ± 19 Hep G2^(b) 2 >200 MDBK 7 36 ± 7BHK-21 2 132 ± 6  H9c2 2 >250^(a)All cytotoxicity testing was done under conditions of rapid celldivision^(b)Cells were grown on collagen coated plates for 4 or 10 dBone Marrow Toxicity Assay

Human bone marrow cells were collected from normal healthy volunteersand the mononuclear population were separated by Ficoll-Hypaque gradientcentrifugation as described previously by Sommadossi J-P, Carlisle R.Antimicrobial Agents and Chemotherapy 1987; 31:452-454; and SommadossiJ-P, Schinazi R F, et al. Biochemical Pharmacology 1992; 44:1921-1925.The culture assays for CFU-GM and BFU-E were performed using a bilayersoft agar or methylcellulose method. Drugs were diluted in tissueculture medium and filtered. After 14 to 18 days at 37° C. in ahumidified atmosphere of 5% CO₂ in air, colonies of greater than 50cells were counted using an inverted microscope. The results arepresented as the percent inhibition of colony formation in the presenceof drug compared to solvent control cultures.

Cell Protection Assay (CPA)

The assay was performed essentially as described by Baginski, S. G.;Pevear, D. PNAS USA 2000, 97(14), 7981-7986. MDBK cells (ATCC) wereseeded onto 96-well culture plates (4,000 cells per well) 24 hoursbefore use. After infection with BVDV (strain NADL, ATCC) at amultiplicity of infection (MOI) of 0.02 plaque forming units (PFU) percell, serial dilutions of test compounds were added to both infected anduninfected cells in a final concentration of 0.5% DMSO in growth medium.Each dilution was tested in quadruplicate. Cell densities and virusinocula were adjusted to ensure continuous cell growth throughout theexperiment and to achieve more than 90% virus-induced cell destructionin the untreated controls after four days post-infection. After fourdays, plates were fixed with 50% TCA and stained with sulforhodamine B.The optical density of the wells was read in a microplate reader at 550nm. The 50% effective concentration (EC₅₀) values were defined as thecompound concentration that achieved 50% reduction of cytopathic effectof the virus.

The myelosuppressive effects of certain nucleoside analogs havehighlighted the need to test for potential effects of investigationaldrugs on the growth of human bone marrow progenitor cells in clonogenicassays. In particular, anemia and neutropenia are the most commondrug-related clinical toxicities associated with the anti-HIV drugzidovudine (ZDV) or the ribavirin (RBV) component of the standard ofcare combination therapy used for HCV treatment. These toxicities havebeen modeled in an in vitro assay that employed bone marrow cellsobtained from healthy volunteers (Sommadossi J-P, Carlisle R.Antimicrob. Agents Chemother. 1987; 31(3): 452-454). ZDV has beenpreviously shown to directly inhibit human granulocyte-macrophagecolony-forming (CFU-GM) and erythroid burst-forming M in this model(BFU-E) activity at clinically relevant concentrations of 1-2 (Berman E,et al. Blood 1989; 74(4):1281-1286; Yoshida Y, Yoshida C. AIDS Res. Hum.Retroviruses 1990; 6(7):929-932.; Lerza R, et al. Exp. Hematol. 1997;25(3):252-255; Domsife R E, Averett D R. Antimicrob. Agents Chemother.1996; 40(2):514-519; Kurtzberg J, Carter S G. Exp. Hematol. 1990;18(10):1094-1096; Weinberg R S, et al. Mt. Sinai J. Med. 1998;65(1):5-13). Using human bone marrow clonogenic assays, the CC₅₀ valuesof mCyd in CFU-GM and BFU-E were 14.1±4.5 and 13.9±3.2 μM (see Table27). mCyd was significantly less toxic to bone marrow cells than bothZDV and RBV (Table 27). TABLE 27 Bone Marrow Toxicity of mCyd inGranulocyte Macrophage Progenitor and Erythrocyte Precursor CellsCFU-GM^(a) BFU-E^(a) Compound CC₅₀ (μM) CC₅₀ (μM) mCyd 14.1 ± 4.5 μM13.9 ± 3.2 ZDV 0.89 ± 0.47 0.35 ± 0.28 RBV 7.49 ± 2.20 0.99 ± 0.24^(a)Data from 3 independent experiments for RBV and 5-8 independentexperiments for mCyd and ZDV. All experiments were done in triplicate.Effect on Mitochondrial Function

Antiviral nucleoside analogs approved for HIV therapy such as ZDV,stavudine (d4T), didanosine (ddI), and zalcitabine (ddC) have beenoccasionally associated with clinically limiting delayed toxicities suchas peripheral neuropathy, myopathy, and pancreatitis (Browne M J, et al.J. Infect. Dis. 1993; 167(1):21-29; Fischl M A, et al. Ann. Intern. Med.1993; 18(10):762-769.; Richman D D, et al. N. Engl. J. Med. 1987;317(4): 192-197; Yarchoan R, et al. Lancet 1990; 336(8714):526-529).These clinical adverse events have been attributed by some experts toinhibition of mitochondrial function due to reduction in mitochondrialDNA (mtDNA) content and nucleoside analog incorporation into mtDNA. Inaddition, one particular nucleoside analog, fialuridine(1,-2′-deoxy-2′-fluoro-1-β-D-arabinofuranosyl-5-iodo-uracil; FIAU),caused hepatic failure, pancreatitis, neuropathy, myopathy and lacticacidosis due to direct mitochondrial toxicity (McKenzie R, et al. N.Engl. J. Med. 1995; 333(17):1099-1105). Drug-associated increases inlactic acid production can be considered a marker of impairedmitochondrial function or oxidative phosphorylation. (Colacino, J. M.Antiviral Res. 1996 29(2-3): 125-39).

To assess the potential of mCyd to produce mitochondrial toxicity,several in vitro studies were conducted using the human hepatoma celllines HepG2 or Huh7. These studies included analysis of lactic acidproduction, mtDNA content, and determination of changes in morphology(e.g., loss of cristae, matrix dissolution and swelling, and lipiddroplet formation) of mitochondrial ultrastructure.

The effects of mCyd on mitochondria are presented in Table 28. Nodifferences were observed in lactic acid production in mCyd-treatedcells versus untreated cells at up to 50 μM mCyd in Huh7 cells or 10 μMmCyd in HepG2 cells. A modest (38%) increase in lactic acid productionwas seen in HepG2 cells treated with 50 μM mCyd. The significance ofthis finding is unclear, particularly since mCyd is unlikely to attain aplasma concentration of 50 μM in the clinic. For comparison, lactic acidproduction increases by 100% over control cells in cells treated with 10μM FIAU (Cui L, Yoon, et al. J. Clin. Invest. 1995; 95: 555-563).Exposure of HepG2 cells to mCyd for 6 or 14 days at concentrations up to50 μM had no negative effect on mitochondrial DNA content compared to a56 or 80% reduction in ddC-treated cells, respectively.

Following M mCyd, the ultrastructure of HepG2 cells, and in □14 days ofexposure to particular mitochondria, was examined by transmissionelectron microscopy. No changes in cell architecture, or inmitochondrial number or morphology (including cristae), were observed inthe majority of cells. In 17% of the cells, 1 to 2 mitochondria out ofan average of 25 per cell appeared enlarged. Such minor changes would beunlikely to have any significant impact on mitochondrial function.ddC-treated cells showed abnormal mitochondrial morphology with loss ofcristae, and the accumulation of fat droplets. (Medina, D. J., C. H.Tsai, et al. Antimicrob. Agents Chemother. 1994 38(8): 1824-8; Lewis W,et al. J. Clin. Invest. 1992; 89(4):1354-1360., Lewis, L. D., F. M.Hamzeh, et al. Antimicrob. Agents Chemother. 1992 36(9): 2061-5). TABLE28 Effect of mCyd on Hepatocyte Proliferation, Mitochondrial Function,and Morphology in HepG2 Cells L-Lactate mtDNA/nuclear DNA ElectronMicroscopy^(c) (% of Control^(a)) (% of Control^(b)) Lipid Conc HepG2Huh7 6 day 14 day Droplet Agent (μM) Cells Cells Treatment TreatmentForm. Mito. Morphol. Cont. 0 100 100 100 100 Negative Normal mCyd 10 98.6 ± 7.3 98.0 ± 12.3 117.3 ± 17.5 99.7 ± 23.9 Negative Normal^(d) 50138.0 ± 8.9 97.1 ± 10.1 158.2 ± 17.5 83.0 ± 15.5 nd nd ddC 1 nd nd 44.3± 9.3 19.6 ± 8.2  nd nd 10 nd nd nd nd Positive Loss of CristaeEffect on Human DNA Polymerases α, β, and γ

The cellular DNA polymerases are responsible for normal nuclear andmitochondrial DNA synthesis and repair. Nucleoside analog triphosphatesare potential inhibitors of DNA polymerases and hence could disruptcritical cell functions. In particular, the inhibition of humanpolymerase γ, the enzyme responsible for mitochondrial DNA synthesis,has been linked to defects in mitochondrial function (Lewis, W., E. S.Levine, et al. Proceedings of the National Academy of Sciences, USA 199693(8): 3592-7.). Experiments were undertaken to determine if mCyd-TPinhibited human DNA polymerases. As shown in Table 29 mCyd-TP was not asubstrate for human DNA polymerases α, β, or γ. Even 1 mM mCyd-TP failedto inhibit these enzymes by 50% in the majority of replicate assays andIC₅₀ values could only be determined to be in excess of 880-1000 μM. Incontrast, ddC was a potent inhibitor of all three human DNA polymerasesand of polymerases β and γ in particular (IC₅₀s of 4.8 and 2.7 μM,respectively). Potent inhibition was also seen for the control drug,actinomycin D, a known inhibitor of DNA-dependent-DNA polymerases. TABLE29 Inhibition of Human Polymerases by mCyd-Triphosphate IC₅₀ (μM)mCyd-TP^(a) ddC-TP^(b) Act. D^(a) Pol α >1000   78 ± 23.4   5.8 ± 3.1Pol β ≧883.3 ± 165 4.8 ± 1 7.9 ± 3 Pol γ ≧929.3 ± 100 2.7 ± 1 15.5 ± 4 ^(a)Mean ± S.D. from 4 data sets^(b)Mean ± S.D. from 2 data sets^(a)HepG2 or huh7 cells were treated with compounds for 4 days, datarepresent at least three independent experiments^(b)HepG2 cells were treated with compounds for 6 and 14 days, datarepresents at least three independent experiments^(c)HepG2 cells were treated with compounds for 14 days^(d)17% cells (11 of 64) contained 1 or 2 enlarged mitochondria out of25 in two independent experimentsnd, not determined

EXAMPLE 28 In Vitro Antiviral Activity Against BVDV

Compounds can exhibit anti-flavivirus or pestivirus activity byinhibiting flavivirus or pestivirus polymerase, by inhibiting otherenzymes needed in the replication cycle, or by other pathways.

Plaque Reduction Assay

For each compound the effective concentration was determined induplicate 24-well plates by plaque reduction assays. Cell monolayerswere infected with 100 PFU/well of virus. Then, serial dilutions of testcompounds in MEM supplemented with 2% inactivated serum and 0.75% ofmethyl cellulose were added to the monolayers. Cultures were furtherincubated at 37° C. for 3 days, then fixed with 50% ethanol and 0.8%Crystal Violet, washed and air-dried. Then plaques were counted todetermine the concentration to obtain 90% virus suppression.

Yield Reduction Assay

For each compound the concentration to obtain a 6-log reduction in viralload was determined in duplicate 24-well plates by yield reductionassays. The assay was performed as described by Baginski, S. G.; Pevear,D. C.; Seipel, M.; et al. PNAS USA 2000, 97(14), 7981-7986, with minormodifications. Briefly, MDBK cells were seeded onto 24-well plates(2×105 cells per well) 24 hours before infection with BVDV (NADL strain)at a multiplicity of infection (MOI) of 0.1 PFU per cell. Serialdilutions of test compounds were added to cells in a final concentrationof 0.5% DMSO in growth medium. Each dilution was tested in triplicate.After three days, cell cultures (cell monolayers and supernatants) werelysed by three freeze-thaw cycles, and virus yield was quantified byplaque assay. Briefly, MDBK cells were seeded onto 6-well plates (5×105cells per well) 24 h before use. Cells were inoculated with 0.2 mL oftest lysates for 1 hour, washed and overlaid with 0.5% agarose in growthmedium. After 3 days, cell monolayers were fixed with 3.5% formaldehydeand stained with 1% crystal violet (w/v in 50% ethanol) to visualizeplaques. The plaques were counted to determine the concentration toobtain a 6-log reduction in viral load.

Studies on the antiviral activity of mCyd in cultured cells wereconducted. The primary assay used to determine mCyd antiviral potencywas a BVDV-based cell-protection assay (CPA). This assay measures theability of mCyd to protect growing MDBK bovine kidney cells fromdestruction by a cytopathic NADL strain of BVDV. The cytotoxicity of thetest drug on uninfected cells was measured in parallel. The antiviralactivities of mCyd and ribavirin in the CPA are compared in Table 30a.mCyd effectively protected de novo-infected MDBK cells in aconcentration-dependent manner with an EC₅₀=0.67±0.22 μM (Table 30a).mCyd afforded complete cytoprotection at concentrations well below theCC₅₀ for mCyd in this assay (38±9 μM). In the CPA, as well as in otherassays described below, ribavirin showed no clear antiviral effect:significant (50% or more) cell protection was not achieved in mostassays as the cytotoxicity of ribavirin overlaps and masks theprotective effect. Thus, ribavirin gave a CC₅₀ of 4.3±0.6 μM and anEC₅₀>4.3 μM in the CPA.

For Tables 30a-30o below, cell lines utilized include MT-4 for HIV; Vero76, African green monkey kidney cells for SARS; BHK for Bovine ViralDiarrhea Virus; Sb-1 for poliovirus Sabin type-1; CVB-2, CVB-3, CVB-4,and CVA-9 for Coxsackieviruses B-2, B-3, B-4 and A-9; and REO-1 fordouble-stranded RNA viruses. Note: BVDV=bovine viral diarrhea virus;YFV=yellow fever virus; DENV=dengue virus; WNV=West Nile virus;CVB-2=Coxsackie B-2 virus; Sb-1=Sabin type 1 poliomyelitis virus; andREO=double-stranded RNA Reovirus. TABLE 30a In Vitro Activity of mCydAgainst BVDV in the Cell Protection Assay Compound n EC₅₀, μM CC₅₀, μMmCyd 11 0.67 ± 0.22 38 ± 9  RBV 3 >4.3 4.3 ± 0.6

TABLE 30b CC₅₀ Test Results for β-D-2′-C-methyl-cytidine (Compound G),3′-O-valinyl ester of β-D-2′-C-methyl-cytidine dihydrochloride salt(Compound M), and β-D-2′-C-methyl-uracil (Compound N) Com- DENV poundCC₅₀ BVDV YFV 2 WNV CVB-2 Sb-1 REO G 34 2.3 54 95 80 12 11.5 13 M 24 5.882 >100 82 12 14 22 N >100 18 100 > or = 80 >100 55 >100 100

TABLE 30c CC₅₀ and EC₅₀ Test Results for β-D-2′-C-methyl-cytidine(Compound G) CC₅₀ CC₅₀ CC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ Compound MT-4Vero 76 BHK Sb-1 CVB-2 CVB-3 CVB-4 CVA-9 REO-1 G 34 >100 >100 6 11 9 1326 13

TABLE 30d CC₅₀ and EC₅₀ Test Results for β-D-2′-C-methyl-adenosine(Compound A) and β-D-2′-C-methyl-2-amino adenosine (Compound B) CC₅₀CC₅₀ CC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ Compound MT-4 Vero 76 BHK Sb-1CVB-2 CVB-3 CVB-4 CVA-9 REO-1 A 4 80 70 10 10 14 13 12 >70 B >100 >10050 90 75 23 32 39 2

TABLE 30e CC₅₀ and EC₅₀ Test Results for β-D-2′-C-methyl-guanosine(Compound C) and β-D-2′-C-methyl-6-chloro-guanosine (Compound D) CC₅₀CC₅₀ CC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ Compound MT-4 Vero 76 BHK Sb-1CVB-2 CVB-3 CVB-4 CVA-9 REO-1 C >100 >100 100 22 30 22 12 46 2D >100 >100 30 50 25 21 25 37 0.4

TABLE 30f CC₅₀ and EC₅₀ Test Results for 3′,5′-di-O-valinyl ester ofβ-D-2′-C-methyl-guanosine dihydrochloride salt (Compound E) CC₅₀ CC₅₀CC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ Compound MT-4 Vero 76 BHK Sb-1 CVB-2CVB-3 CVB-4 CVA-9 REO-1 E >100 >100 100 30 33 30 35 40 2

TABLE 30g CC₅₀ and EC₅₀ Test Results for β-D-2′-C-methyl-cytidine(Compound G) CC₅₀ CC₅₀ CC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ Compound MT-4Vero 76 BHK Sb-1 CVB-2 CVB-3 CVB-4 CVA-9 REO-1 G 34 >100 >100 6 11 9 1326 13

TABLE 30h CC₅₀ and EC₅₀ Test Results for β-D-2′-C-ethynyl-adenosine(Compound H) CC₅₀ CC₅₀ CC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ Compound MT-4Vero 76 BHK Sb-1 CVB-2 CVB-3 CVB-4 CVA-9 REO-1 H 4.6 60 15 1 1.5 1 2 2.56

TABLE 30i CC₅₀ and EC₅₀ Test Results for β-D-2′-C-ethynyl-cytidine(Compound I) CC₅₀ CC₅₀ CC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ Compound MT-4Vero 76 BHK Sb-1 CVB-2 CVB-3 CVB-4 CVA-9 REO-1 I > or = >100 >100 26 3333 24 59 >100 100

TABLE 30j CC₅₀ and EC₅₀ Test Results for β-D-2-amino-adenosine (CompoundJ) CC₅₀ CC₅₀ CC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ EC₅₀ Compound MT-4 Vero 76BHK Sb-1 CVB-2 CVB-3 CVB-4 CVA-9 REO-1 J 50 >100 >100 40 53 55 50 53>100

TABLE 30k CC₅₀ Test Results for β-D-2′-C-methyl-adenosine (Compound A),β-D-2′-C-methyl-2-amino adenosine (Compound B), andβ-D-2′-C-methyl-2-amino-6-cyclopropyl adenosine(Compound K) Com- DENVCVB- pound CC₅₀ BVDV YFV 2 WNV 2 Sb-1 REO A 4.0 1.2 2.7 2.7 3.6 7 7 >70B >100 2.1 0.8 0.7 0.3 76 90 2 K >100 18 10 4.9 3.5 >100 >100 9.5

TABLE 30l CC₅₀ Test Results for β-D-2′-C-methyl-guanosine (Compound C),β-D-2′-C-methyl-1-(methyl-2-oxo-2-phenyl ethyl)guanosine (Compound L),and β-D-2′-C-methyl-6-chloro guanosine (Compound D) Com- DENV pound CC₅₀BVDV YFV 2 WNV CVB-2 Sb-1 REO C >100 3.5 1.2 1.4 0.6 29 50 2 L >100 12 64.4 3 >100 >100 12 D >100 0.7 1.0 0.7 0.3 25 50 0.4

TABLE 30m CC₅₀ Test Results for 3′,5′-di-O-valinyl ester ofβ-D-2′-C-methyl-guanosine dihydrochloride salt (Compound E) Com- DENVpound CC₅₀ BVDV YFV 2 WNV CVB-2 Sb-1 REO E >100 4.9 1.0 1.4 1 33 55 2.1

TABLE 30n CC₅₀ Test Results for β-D-2′-C-ethynyl-adenosine (Compound H)Com- DENV pound CC₅₀ BVDV YFV 2 WNV CVB-2 Sb-1 REO H 4.6 0.4 2.0 1.1 11.2 0.7 6

TABLE 30o CC₅₀ Test Results for β-D-2′-C-methyl-cytidine (Compound G),3′-O-valinyl ester of β-D-2′-C-methyl-cytidine dihydrochloride salt(Compound M), and β-D-2′-C-methyl-uracil (Compound N) Com- DENV poundCC₅₀ BVDV YFV 2 WNV CVB-2 Sb-1 REO G 34 2.3 54 95 80 12 11.5 13 M 24 5.882 >100 82 12 14 22 N >100 18 100 > or = 80 >100 55 >100 100

The overall antiviral potency of mCyd was determined against differentstrains of BVDV and both cytopathic (cp) and noncytopathic (ncp)biotypes in cell protection assays as well as in plaque reduction andyield reduction assays. The latter assays measure the output ofinfectious virus from cells and hence provide a stringent test ofantiviral efficacy. The different data sets from all three assays showagreement as summarized in Table 31. The range of 50% and 90% effectiveinhibitory concentration (EC₅₀ and EC₉₀) values for mCyd was 0.3 to 2.8μM and 0.87 to 4.8 μM, respectively.

In the BVDV yield reduction assay, subcytotoxic concentrations (circa 20μM) of mCyd suppressed de novo BVDV production by up to 6 logs, to thepoint where no infectious virus was detected. A 4 log₁₀ effectivereduction in BVDV production (EC_(4log10) or EC_(99.99)) was attainedbetween 6.0 and 13.9 μM mCyd. In contrast, interferon alpha 2b (IFNα2b), although active against BVDV in this assay (EC₅₀ 2.6 IU per ml),never gave more than 2 logs of viral reduction, even at 1000 IU per ml.Thus, the antiviral effect of mCyd against BVDV was much greater thanthat of IFNα2b or RBV.

EXAMPLE 29 In Vitro Antiviral Activity Against other Positive-Strand RNAViruses

mCyd has been tested for efficacy against positive-strand RNA virusesother than BVDV. Data obtained are summarized in Table 31 and 32.Against flaviruses, mCyd showed modest activity. The composite EC₅₀ranges (in μM) determined from both sites were: West Nile virus (46-97);Yellow Fever virus (9-80); and Dengue Virus (59-95). For mCyd againstthe alpha virus, Venezuelan Equine Encephalitis virus, EC₅₀ values were1.3-45 μM. mCyd was broadly active against Picomoviruses, such as Poliovirus (EC₅₀=6 μM), Coxsackie virus (EC₅₀=15 μM), Rhinovirus types 5 and14 (EC₅₀s=<0.1 and 0.6 μg/ml) and Rhinovirus type 2 (EC₅₀ 2-10 μM). mCydwas generally inactive against all RNA and DNA viruses tested except forthe positive-strand RNA viruses. mCyd was also found to have no activityagainst HIV in MT-4 human T lymphocyte cells or HBV in HepG2.2.15 cells.TABLE 31 In Vitro Antiviral Activity of mCyd Against Plus-Strand RNAViruses Method of Cell Antiviral Efficacy (μM) Assay Virus Type Type nEC₅₀ EC₉₀ EC_(4 log) Cell Protection Assay BVDV NADL cp MDBK 11 0.67 ±0.22 Yield Reduction Assay BVDV NADL cp MDBK 3 2.77 ± 1.16  4.8 ± 1.5513.9 ± 3.07 BVDV New York-1 ncp MDBK 6 0.30 ± 0.07 0.87 ± 0.18 6.03 ±1.41 BVDV I-NADL cp MDBK 1 0.68 1.73 8.22 BVDV I-N-dIns ncp MDBK 1 0.591.49 7.14 Plaque Reduction Assay BVDV NADL cp MDBK 3 2.57 ± 0.35 4.63 ±0.72 Cell Protection Assay West Nile Virus BHK 3 63-97 Cell ProtectionAssay Yellow Fever Virus 17D BHK 1 60-80 DENV-2 BHK 2 95 Cell ProtectionAssay DENV-4 BHK 1 59 Polio Virus Plaque Reduction Assay Sb-1 VERO 1 6Plaque Reduction Assay Coxsackie Virus B2 VERO 1 15cp, cytopathic virus; ncp noncytopathic virus1-NADL cp and I-N-dIns ncp represent recombinant BVDV viruses

TABLE 32 In Vitro Antiviral Activity, Selectivity, and Cytotoxicity ofmCyd Virus (Cell line)^(a) EC₅₀ ^(b)(μM) CC₅₀ ^(c)(μM) WNV (Vero) 46114-124 YFV (Vero) 9-30  150->200 VEE (Vero) 1.3-45   >200 HSV-1(HFF)^(d) >100 >100 HSV-2 (HFF)^(d) >100 >100 VZV (HFF)^(d) >20 67.8 EBV(Daudi)^(d) 25.5 >50 HCMV (HFF)^(d) 9.9-15.6 67-73 MCMV (MEF) >0.8 2.4Influenza A/H1N1 (MDCK) >200 >200 Influenza A/H3N2 (MDCK) >20 45-65Influenza B (MDCK) >200  55-140 Adenovirus type 1 (A549) >200 >200Parainfluenza type 3 (MA-104) >200 >200 Rhinovirus type 2 (KB) 2-10 >200Rhinovirus type 5 (KB)^(d) 0.6 20-30 Rhinovirus type 14 (HeLa-Ohio)^(d)<0.1  20->100 RSV type A (MA-104) >200 200 Punta Toro A (LLC-MK2) >200>200^(a)HFF, human foreskin fibroblast; Daudi, Burkitt's B-cell lymphoma;MDCK, canine kidney cells; CV-1, African green monkey kidney cells; KB,human nasopharyngeal carcinoma; MA-104, Rhesus monkey kidney cells;LLC-MK2, Rhesus monkey kidney cells; A549, Human lung carcinoma cells;MEF, mouse embryo fibroblast; Vero, African green monkey kidney cells;HeLa, human cervical adenocarcinoma cells.^(b)EC₅₀ = 50% effective concentration.^(c)CC₅₀ = 50% cytotoxic concentration.^(d)Result presented in μg/mL rather than μM.

EXAMPLE 30 Multiplicity of Infection (MOI) and Antiviral Efficacy

The cell protection assay format was used to test the effect ofincreasing the amount of BVDV virus on the EC₅₀ of mCyd. Increasing theMOI of BVDV in this assay from 0.04 to 0.16, caused the EC₅₀ of mCyd toincrease linearly from 0.5 μM to approximately 2.2 μM.

EXAMPLE 31 Viral Rebound in mCyd Treated Cells

The effect of discontinuing treatment with mCyd was tested in MDBK cellspersistently infected with a noncytopathic strain (strain I-N-dIns) ofBVDV. Upon passaging in cell culture, these cells continuously produceanywhere from 10⁶ to >10⁷ infectious virus particles per ml of media.This virus can be measured by adding culture supernatants from treatedMDBK (BVDV) cells to uninfected MDBK cells and counting the number ofresultant viral foci after disclosure by immunostaining with aBVDV-specific antibody. Treatment of a persistently infected cell linewith 4 μM mCyd for one cell passage (3 days) reduced the BVDV titer byapproximately 3 log₁₀ from pretreatment and control cell levels of justunder 10⁷ infectious units per ml. At this point, mCyd treatment wasdiscontinued. Within a single passage, BVDV titers rebounded tountreated control levels of just over 10⁷ infectious units per ml.

EXAMPLE 32 Mechanism of Action

In standard BVDV CPA assays, mCyd treatment results in a marked increasein total cellular RNA content as cells grow, protected from thecytopathic effects of BVDV. This was coupled with a marked decrease inthe production of BVDV RNA due to mCyd. Conversely, in the absence ofmCyd, total cellular RNA actually decreases as BVDV RNA rises due to thedestruction of the cells by the cytopathic virus. To further test theeffect of mCyd on viral and cellular RNAs, the accumulation ofintracellular BVDV RNA was monitored in MDBK cells 18-hours postinfection (after approximately one cycle of virus replication) usingReal Time RT-PCR. In parallel, a cellular housekeeping ribosomal proteinmRNA (rig S 15 mRNA) was also quantitated by RT-PCR using specificprimers. The results showed that mCyd dramatically reduced BVDV RNAlevels in de novo-infected MDBK cells with an EC₅₀ of 1.7 μM and an EC₉₀of 2.3 μM. The maximum viral RNA reduction was 4 log₁₀ at the highestinhibitor concentration tested (125 μM). No effect on the level of therig S15 cellular mRNA control was observed. Together, the precedingfindings suggest that mCyd inhibits BVDV by specifically interferingwith viral genome RNA synthesis without impacting cellular RNA content.This idea is further supported by the observation (Table 26a) thatinhibition of RNA synthesis as measured by [³1H]-uridine uptake in HepG2cells requires high concentrations of mcyd (EC₅₀=186 μM).

In in vitro studies using purified BVDV NS5B RNA-dependent RNApolymerase (Kao, C. C., A. M. Del Vecchio, et al. 1999. Virology 253(1):1-7) and synthetic RNA templates, mCyd-TP inhibited RNA synthesis withan IC₅₀ of 0.74 μM and was a competitive inhibitor of BVDV NS5BRNA-dependent RNA polymerase with respect to the natural CTP substratE.The inhibition constant (K_(i)) for mCyd-TP was 0.16 μM and theMichaelis-Menten constant (K_(m)) for CTP was 0.03 μM. Inhibition of RNAsynthesis by mCyd-TP required the presence of a cognate G residue in theRNA template. The effect of mCyd-TP on RNA synthesis in the absence ofCTP was investigated in more detail using a series of short (21mer)synthetic RNA templates containing a single G residue, which was movedprogressively along the template. Analysis of the newly synthesizedtranscripts generated from these templates in the presence of mCyd-TPrevealed that RNA elongation continued only as far as the G residue,then stopped (FIG. 12). In templates containing more than one G residue,RNA synthesis stopped at the first G residue encountered by thepolymerase. These data strongly suggest that m-Cyd-TP is acting as anon-obligate chain terminator. The mechanism of this apparent chaintermination is under further investigation.

EXAMPLE 33 Eradication of A Persistent Bvdv Infection

The ability of mCyd to eradicate a viral infection was tested in MDBKcells persistently infected with a noncytopathic strain of BVDV (strainI-N-dIns). (Vassilev, V. B. and R. O. Donis Virus Res. 2000 69(2):95-107.) Compared to untreated cells, treatment of persistently infectedcells with 16 μM mCyd reduced virus production from more than 6 logs ofvirus per ml to undetectable levels within two cell passages (3 to 4days per passage). No further virus production was seen upon continuedtreatment with mCyd through passage 12. At passages 8, 9 and 10 (arrows,FIG. 13), a portion of cells was cultured for two further passages inthe absence of drug to give enough time for mCyd-TP to decay and virusreplication to resume. The culture media from the cells were repeatedlytested for the re-emergence of virus by adding culture supernatants fromtreated MDBK (BVDV) cells to uninfected MDBK cells and counting theresultant viral foci after disclosure by immunostaining with aBVDV-specific antibody. Although this assay can detect a single virusparticle, no virus emerged from the cells post drug treatment. Thus,treatment with mCyd for 8 or more passages was sufficient to eliminatevirus from the persistently infected cells.

EXAMPLE 34 Combination Studies with Interferon Alpha 2B

The first study, performed in MDBK cells persistently infected with theNew York-1 (NY-1) strain of BVDV, compared the effect of monotherapywith either mCyd (8 μM) or interferon alpha 2b (200 IU/ml), or the twodrugs in combination (FIG. 14A). In this experiment, 8 μM mCyd alonereduced viral titers by approximately 3.5 log₁₀ after one passage to alevel that was maintained for two more passages. Interferon alpha 2balone was essentially inactive against persistent BVDV infection(approximately 0.1 log₁₀ reduction in virus titer) despite being activeagainst de novo BVDV infection. However, the combination of mCyd plusinterferon alpha 2b reduced virus to undetectable levels by the secondpassage and clearly showed better efficacy to either monotherapy.

In a follow up study (FIG. 14B) of MDBK cells persistently infected withthe I-N-dIns noncytopathic strain of BVDV, mCyd was supplied at fixeddoses of 0, 2, 4 and 8 μM, while interferon alpha 2b was titrated from 0to 2,000 IU per ml. Again, interferon alpha 2b was essentially inactive(0.1 log reduction in viral titer), while mCyd alone inhibited BVDV(strain I-N-dins) propagation in a dose-dependent manner. mCyd at 8 μMreduced virus production by 6.2 log₁₀, to almost background levels.

EXAMPLE 35 Resistance Development

In early cell culture studies, repeated passaging of a cytopathic strainof BVDV in MDBK cells in the presence of mCyd failed to generateresistant mutants, suggesting that the isolation mCyd-resistant BVDVmutants is difficult. However, studies in cell lines persistentlyinfected with noncytopathic forms of BVDV led to the selection ofresistant virus upon relatively prolonged treatment with mCyd atsuboptimal therapeutic concentrations of drug (2 to 8 μM, depending onthe experiment). In the representative experiment shown in FIG. 15A, thevirus was no longer detectable after two passages in the presence of 8μM mCyd, but re-emerged by passage 6. The lower titer of the re-emergentvirus is apparent from the data: resistant virus typically has a 10 foldor more lower titer than the wild-type virus and was easily suppressedby co-therapy with intronA (FIG. 15A). The phenotype of the virus thatre-emerged was remarkably different from the initial wild-type virus: asshown in FIG. 15B, it yielded much smaller foci (typically, 3 to 10times smaller in diameter then those of the wild-type virus). Thisphenotype did not change after prolonged passaging in culture in thepresence of the inhibitor (at least 72 days), however, it quicklyreverted to the wild-type phenotype (large foci) after thediscontinuation of the treatment.

RT-PCR sequencing of the resistant mutant was used to identify themutation responsible for resistance. Sequencing efforts were focused onthe NS5B RNA-dependent RNA polymerase region of BVDV, which was assumedto be the likely target for a nucleoside inhibitor. A specific S405Tamino-acid substitution was identified at the start of the highlyconserved B domain motif of the polymerase. The B domain is part of thepolymerase active site and is thought to be involved in nucleosidebinding (Lesburg, C. A., M. B. Cable, et al. Nature Structural Biology1999 6(10): 937-43). Resistance to nucleosides has been mapped to thisdomain for other viruses such as HBV (Ono et al, J. Clin. Invest. 2001February; 107(4):449-55.). To confirm that this mutation was responsiblefor the observed resistance, the mutation was reintroduced into thebackbone of a recombinant molecular clone of BVDV. The resulting clonewas indistinguishable in phenotypic properties from the isolated mutantvirus, confirming that the S405T mutation is responsible for resistanceand that the NS5B RNA-dependent RNA polymerase is the molecular targetfor mCyd. The highly conserved nature of this motif at the nucleotidesequence (Lai, V. C., C. C. Kao, et al. J. Virol. 1999 73(12): 10129-36)and structural level among positive-strand RNA viruses (including HCV)allows a prediction that the equivalent mutation in the HCV NS5BRNA-dependent RNA polymerase would likely be S282T.

S405T mutant BVDV was refractory to mCyd up to the highestconcentrations that could be tested (EC₅₀>32 μM), but was alsosignificantly impaired in viability compared to wild-type virus. Asnoted above, the S405T mutant exhibited a 1-2 log₁₀ lower titer thanwild-type. BVDV and produced much smaller viral plaques. In addition,the mutant virus showed a marked reduction in the rate of a single cycleof replication (>1000-fold lower virus titer at 12 h), and accumulatedto about 100 fold lower levels than the wild-type virus even after 36 hof replication (FIG. 15C). The virus also quickly reverted to wild-typevirus upon drug withdrawal. Finally, the mutant was also more sensitive(˜40 fold) to treatment with IFN alpha 2b than wild-type as shown inFIG. 15D.

A second, additional mutation, C446S, was observed upon furtherpassaging of the S405T mutant virus in the presence of drug. Thismutation occurs immediately prior to the essential GDD motif in the Cdomain of BVDV NS5B RNA-dependent RNA polymerase.

Preliminary studies suggest that a virus bearing both mutations does notreplicate significantly better than the S405T mutant, hence thecontribution of this mutation to viral fitness remains unclear. Furtherstudies to characterize resistance development are ongoing.

EXAMPLE 36 In Vivo Antiviral Activity of Val-mCyd in an Animal EfficacyModel

Chimpanzees chronically infected with HCV are the most widely acceptedanimal model of HCV infection in human patients (Lanford, R. E., C.Bigger, et al. Ilar J. 2001 42(2): 117-26; Grakoui, A., H. L. Hanson, etal. Hepatology 2001 33(3): 489-95). A single in vivo study of the oraladministration of val-mCyd in the chimpanzee model of chronic hepatitisC virus infection has been conducted.

HCV genotyping on the five chimpanzees was performed by the SouthwestFoundation Primate Center as part of their mandated internal Health andMaintenance Program, designed to ascertain the disease status of allanimals in the facility to identify potential safety hazards toemployees. The five chimpanzees used in this study exhibited a high HCVtiter in a genotyping RT PCR assay that distinguishes genotype 1 HCVfrom all other genotypes, but does not distinguish genotype 1a from 1b.This indicates that the chimpanzees used in this study were infectedwith genotype 1 HCV (HCV-1). TABLE 33 Summary of Val-mCyd In VivoActivity Study in the Chimpanzee Model of Chronic HCV InfectionFrequency/ Species Val-mCyd Doses Route of Study Description (N) (mg/kg)(n) Administration Study Endpoints One-week antiviral Chimpanzee 10 and20 (2 each) QD × 7 days Serum HCV RNA, serum activity of mCyd in (5)[equivalent to 8.3 (PO) chemistries, CBCs, chronically hepatitis C and16.6 mpk of general well being, and virus (genotype 1)- free base], andclinical observations infected chimpanzees vehicle control (1)Seven-Day Antiviral Activity Study in the Chimpanzee Model of ChronicHepatitis C Virus Infection

Four chimpanzees (2 animals per dose group at 10 mg/kg/day or 20mg/kg/day) received val-mCyd dihydrochloride, freshly dissolved in aflavorful fruit drink vehicle. These doses were equivalent to 8.3 and16.6 mg/kg/day of the val-mCyd free base, respectively. A fifth animaldosed with vehicle alone provided a placebo control. The study designincluded three pretreatment bleeds to establish the baseline fluctuationof viral load and three bleeds during the one week of treatment (on days2, 5 and 7 of therapy) to evaluate antiviral efficacy. The analysis wascompleted at the end of the one-week dosing period, with no furtherfollow up.

HCV RNA Determination

Serum levels of HCV RNA throughout the study were determinedindependently by two clinical hospital laboratories. HCV RNA was assayedusing a quantitative RT-PCR nucleic acid amplification test (RocheAmplicor HCV Monitor Test, version 2.0). This assay has a lower limit ofdetection (LLOD) of 600 IU/mL and a linear range of 600-850,000 IU/mL.

To aid in interpretation of the viral load declines seen during therapy,emphasis was placed on determining (i) the extent of fluctuations inbaseline HCV viral load in individual animals, and (ii) the inherentvariability and reproducibility of the HCV viral load assay. To addressthese issues, full viral load data sets obtained from the twolaboratories were compared. The results from both sites were found to beclosely comparable and affirmed both the stability of the pretreatmentHCV viral loads as well as the reliability of the HCV Roche Amplicorassay. To present the most balanced view of the study, the mean valuesderived by combining both data sets were used to generate the resultspresented in FIGS. 16 and 17. FIG. 16 presents the averaged data fordose cohorts, while FIG. 17 presents the individual animal data. Thechanges in viral load from baseline seen during therapy for each animalat each site are also summarized in Table 34.

The HCV viral load analysis from the two sites revealed thatpretreatment HCV viral loads were (i) very similar among all fiveanimals and all 3 dose groups, and (ii) very stable over the 3-weekpretreatment period. The mean pretreatment log₁₀ viral load and standarddeviations among the five individual animals were 5.8±0.1 (site 1) and5.6±0.1 (site 2). These data indicate that the c.v. (coefficient ofvariance) of the assay is only around 2% at both sites. The largestfluctuation in HCV viral load seen in any animal during pretreatment wasapproximately 0.3 log₁₀.

As seen in FIGS. 16 and 17, once a day oral delivery of val-mCydproduced a rapid antiviral effect that was not seen for the placeboanimal, nor during the pretreatment period. Viral titers weresubstantially reduced from baseline after two days of therapy for allanimals receiving val-mCyd, and tended to fall further under continuedtherapy in the two treatment arms. By the end of treatment (day 7), themean reductions from baseline HCV viral load were 0.93 log₁₀ and 1.05log₁₀ for the 8.3 and 16.6 mg/kg/day dose groups, respectively. Thetiter of the placebo animal remained essentially unchanged from baselineduring the therapy period.

An analysis of the data from the two quantification sites on the changesin baseline HCV viral load in response to therapy is presented in Table34. Overall, the two data sets agree well, confirming the reliability ofthe assay. With the exception of animal 501, the difference in viralload between the two sites was generally 0.3 log₁₀ or less, similar tothe fluctuation observed during the pretreatment period. For animal 501,the discrepancy was closer to 0.5 log₁₀. The viral load drop seen inresponse to therapy varied from 0.436 (animal 501, site 1) to 1.514log₁₀ (animal 497, site 2). The latter corresponds to a change in HCVviral load from 535,000 (pretreatment) to 16,500 (day 7) genomes per ml.TABLE 34 Summary of Changes in Baseline Log₁₀ HCV RNA Viral Load DuringTherapy Dose (mpk) Animal ID Site Day 2 Day 5 Day 7 0 499 1 −0.00041−0.11518 0.14085 2 −0.06604 0.10612 −0.16273 8.3 500 1 −1.15634 −0.40385−0.80507 2 −1.07902 −0.55027 −1.06259 8.3 501 1 −0.25180 −0.36179−0.43610 2 −0.45201 −0.71254 −0.90034 16.6 497 1 −0.72148 −0.90704−1.27723 2 −0.85561 −1.01993 −1.51351 16.6 498 1 −0.29472 −0.28139−0.60304 2 −0.65846 −0.55966 −0.69138Exposure of Chimpanzees to mCyd

Limited HPLC analyses were preformed to determine the concentration ofmCyd attained in the sera of chimpanzees following dosing with val-mCyd.In sera drawn 1 to 2 hours post dose on days 2 and 5 of dosing, mCydlevels were typically between 2.9 and 12.1 μM (750 and 3100 ng/mL,respectively) in treated animals. No mCyd was detected in pretreatmentsera or in the placebo control sera. Within 24 hours of the final dose,serum levels of mCyd had fallen to 0.2 to 0.4 μM (50 and 100 ng/mL,respectively). No mUrd was detected in any sera samples although themethodology used has a lower limit of quantification of 0.4 μM (100ng/mL) for mUrd.

Safety of mCyd in the Chimpanzee Model of Chronic HCV Infection

Chimpanzees were monitored by trained veterinarians throughout the studyfor weight loss, temperature, appetite, and general well being, as wellas for blood chemistry profile and CBCs. No adverse events due to drugwere noted. The drug appeared to be well tolerated by all four treatedanimals. All five animals lost some weight during the study and showedsome aspartate aminotransferase (AST) elevations, but these are normaloccurrences related to sedation procedures used, rather than study drug.A single animal experienced an alanine aminotransferase (ALT) flare inthe pretreatment period prior to the start of dosing, but the ALT levelsdiminished during treatment. Thus, this isolated ALT event was notattributable to drug.

EXAMPLE 37 In Vitro Metabolism

Studies were conducted to determine the stability of val-mCyd and mCydin human plasma. Val-mCyd was incubated in human plasma at 0, 21 or 37°C. and samples analyzed at various time points up to 10 hours (FIG. 18).At 37° C., val-mCyd was effectively converted to mCyd, with only 2% ofthe input val-mCyd remaining after 10 hours. The in vitro half-life ofval-mCyd in human plasma at 37° C. was 1.81 hours. In studies of the invitro stability of mCyd in human plasma, or upon treatment with a crudepreparation enriched in human cytidine/deoxycytidine deaminase enzymes,mCyd remained essentially unchanged and no deamination to the uridinederivative of mCyd (mUrd) occurred after incubation at 37° C. Only inrhesus and cynomologus monkey plasma was limited deamination observed.Incubation of mCyd at 37° C. in cynomologus monkey plasma yielded 6.7and 13.0% of mUrd deamination product after 24 and 48 hours,respectively, under conditions where control cytidine analogs wereextensively deaminated.

In addition to the TP derivatives of mCyd and mUrd, minor amounts ofmCyd-5′-diphosphate, mCyd-DP, roughly 10% the amount of thecorresponding TP, were seen in all three cell types. Lesser amounts ofmUrd-DP were detected only in two cell types (primary human hepatocytesand MDBK cells). No monophosphate (MP) metabolites were detected in anycell type. There was no trace of any intracellular mUrd and no evidencefor the formation of liponucleotide metabolites such as the5′-diphosphocholine species seen upon the cellular metabolism of othercytidine analogs.

FIG. 19 shows the decay profiles of mCyd-TP determined followingexposure of HepG2 cells to 10 μM [³H]-mCyd for 24 hours. The apparentintracellular half-life of the mCyd-TP was 13.9±2.2 hours in HepG2 cellsand 7.6±0.6 hours in MDBK cells: the data were not suitable forcalculating the half life of mUrd-TP. The long half life of mCyd-TP inhuman hepatoma cells supports the notion of once-a-day dosing forval-mCyd in clinical trials for HCV therapy. Phosphorylation of mCydoccurred in a dose-dependent manner up to 50 μM drug in all three celltypes, as shown for HepG2 cells in FIG. 19C. Other than the specificdifferences noted above, the phosphorylation pattern detected in primaryhuman hepatocytes was qualitatively similar to that obtained using HepG2or MDBK cells.

Contribution of mUrd

In addition to the intracellular active moiety, mCyd-TP, cells fromdifferent species have been shown to produce variable and lesser amountsof a second triphosphate, mUrd-TP, via deamination of intracellular mCydspecies. The activity of mUrd-TP against BVDV NS5B RNA-dependent RNApolymerase has not been tested to date but is planned. To date, datafrom exploratory cell culture studies on the antiviral efficacy andcytotoxicity of mUrd suggest that mUrd (a) is about 10-fold less potentthan mCyd against BVDV; (b) has essentially no antiviral activityagainst a wide spectrum of other viruses; and (c) is negative whentested at high concentrations in a variety of cytotoxicity tests(including bone marrow assays, mitochondrial function assays andincorporation into cellular nucleic acid). Based on these results, itappears that the contribution of mUrd to the overall antiviral activityor cytotoxicity profile of mCyd is likely to be minor. Extensivetoxicology coverage for the mUrd metabolite of mCyd exists fromsubchronic studies conducted with val-mCyd in the monkey.

EXAMPLE 38 Cellular Pathways for Metabolic Activation

The nature of the enzyme responsible for the phosphorylation of mCyd wasinvestigated in substrate competition experiments. Cytidine (Cyd) is anatural substrate of cytosolic uridine-cytidine kinase (UCK), thepyrimidine salvage enzyme responsible for conversion of Cyd toCyd-5′-monophosphate (CMP). The intracellular phosphorylation of mCyd tomCyd-TP was reduced in the presence of cytidine or uridine in adose-dependent fashion with EC₅₀ values of 19.17±4.67 μM for cytidineand 20.92±7.10 μM for uridine. In contrast, deoxycytidine, a substratefor the enzyme deoxycytidine kinase (dCK), had little effect on theformation of mCyd-TP with an EC₅₀>100 μM. The inhibition of mCydphosphorylation by both cytidine and uridine, but not deoxycytidine,suggests that mCyd is phosphorylated by the pyrimidine salvage enzyme,uridine-cytidine kinase (Van Rompay, A. R., A. Norda, et al. MolPharmacol 2001 59(5): 1181-6). Further studies are required to confirmthe proposed role of this kinase in the activation of mCyd.

EXAMPLE 39 Pathways for the Cellular Biosynthesis of mUrd-TP

As outlined above, mUrd-TP is a minor metabolite arising to varyingextents in cells from different species. mUrd does not originate viaextracellular deamination of mCyd since mUrd is not seen in the cellmedium which also lacks any deamination activities. The cellularmetabolism data are consistent with the idea that mUrd-TP arises via thebiotransformation of intracellular mCyd species. Consideration of theknown ribonucleoside metabolic pathways suggests that the most likelyroutes involve deamination of one of two mCyd species by two distinctdeamination enzymes: either mCyd-MP by a cytidylate deaminase (such asdeoxycytidylate deaminase, dCMPD), or of mCyd by cytidine deaminase(CD). Further phosphorylation steps lead to mUrd-TP. These possibilitiesare under further investigation.

EXAMPLE 40 Clinical Evaluation of Val-mCyd

Patients who met eligibility criteria were randomized into the study atBaseline (Day 1), the first day of study drug administration. Eachdosing cohort was 12 patients, randomized in a 10:2 ratio to treatmentwith drug or matching placebo. Patients visited the study center forprotocol evaluations on Days 1, 2, 4, 8, 11, and 15. After Day 15, studydrug was stopped. Thereafter, patients attended follow-up visits on Days16, 17, 22, and 29. Pharmacokinetic sampling was performed on the firstand last days of treatment (Day 1 and Day 15) on all patients, underfasting conditions.

The antiviral effect of val-mCyd was assessed by (i) the proportion ofpatients with a ≧1.0 log₁₀ decrease from baseline in HCV RNA level atDay 15, (ii) the time to a ≧1.0 log₁₀ decrease in serum HCV RNA level,(iii) the change in HCV RNA level from Day 1 to Day 15, (iv) the changein HCV RNA level from Day 1 to Day 29, (v) the proportion of patientswho experience return to baseline in serum HCV RNA level by Day 29, and(vi) the relationship of val-mCyd dose to HCV RNA change from Day 1 toDay 15.

Clinical Pharmacokinetics of mCyd after Oral Administration ofEscalating Doses of Val-mCyd

Pharmacokinetics were evaluated over a period of 8 h after the firstdose on day 1 and after the last dose on day 15, with 24-h trough levelsmonitored on days 2, 4, 8, 11 and 16, and a 48-h trough on day 17.Plasma concentrations of mCyd, mUrd and Val-mCyd were measured by aHPLC/MS/MS methodology with a lower limit of quantitation (LOQ) at 20ng/ml.

The pharmacokinetics of mCyd was analyzed using a non-compartmentalapproach. As presented in the tables below, the principalpharmacokinetic parameters were comparable on day 1 and day 15,indicative of no plasma drug accumulation after repeated dosing. Theplasma exposure also appears to be a linear function of dose. As shownin the tables below, principal pharmacokinetic parameters of drugexposure (Cmax and AUC) doubled as doses escalated from 50 to 100 mg.TABLE 35 Pharmacokinetic parameters of mCyd at 50 mg C_(max) T_(max)AUC_(0-inf) t_(1/2) Parameters (ng/ml) (h) (ng/ml × h) (h) Day 1 Mean428.1 2.5 3118.7 4.1 SD 175.5 1.1 1246.4 0.6 CV % 41.0 43.2 40.0 13.8Day 15 Mean 362.7 2.2 3168.4 4.6 SD 165.7 1.0 1714.8 1.3 CV % 45.7 46.954.1 28.6

TABLE 36 Pharmacokinetic parameters of mCyd at 100 mg C_(max) T_(max)AUC_(0-inf) t_(1/2) Parameters (ng/ml) (h) (ng/ml × h) (h) Day 1 Mean982.1 2.6 6901.7 4.4 SD 453.2 1.0 2445.7 1.1 CV % 46.1 36.2 35.4 25.2Day 15 Mean 1054.7 2.0 7667.5 4.2 SD 181.0 0.0 1391.5 0.5 CV % 17.2 0.018.1 11.7

The mean day 1 and day 15 plasma kinetic profiles of mCyd at 50 and 100mg are depicted in the FIG. 20.

In summary, following oral administration of val-mCyd, the parentcompound mCyd was detectable in the plasma of HCV-infected subjects.mCyd exhibits linear plasma pharmacokinetics in these subjects acrossthe two dose levels thus far examined. There was no apparentaccumulation of mCyd in subjects' plasma following 15 days of dailydosing at the doses thus far examined.

Antiviral Activity of mCyd after Oral Administration of Escalating Dosesof Val-mCyd Starting at 50 mg/day for 15 Days in HCV-Infected Patients

Serum HCV RNA level were determined with the use of the Amplicor HCVMonitor™ assay v2.0 (Roche Molecular Systems, Branchburg, N.J., USA),which utilizes polymerase chain reaction (PCR) methods. The lower limitof quantification (LLOQ) with this assay was estimated to beapproximately 600 IU/mL and the upper limit of quantification (ULOQ)with this assay was estimated to be approximately 500,000 IU/mL.

Serum samples for HCV RNA were obtained at screening (Day −42 to −7) todetermine eligibility for the study. The Screening serum HCV RNA valuesmust be ≧5 log₁₀ IU/mL by the Amplicor HBV Monitor™ assay at the centralstudy laboratory.

During the study period, serum samples for HCV RNA were obtained atBaseline (Day 1), and at every protocol-stipulated post-Baseline studyvisit (Days 2, 4, 8, 11, 15, 16, 17, 22, and 29). Serum samples for HCVRNA were also collected during protocol-stipulated follow-up visits forpatients prematurely discontinued from the study.

The antiviral activity associated with the first two cohorts (50 and 100mg per day) in the ongoing study is summarized in the following tablesand graphs. Although the duration of dosing was short (15 days) and theinitial dose levels low, there were already apparent effects on thelevels of HCV RNA in the plasma of infected patients. TABLE 37 SummaryStatistics of HCV RNA in Log₁₀ Scale Day Treatment −1 1 2 4 8 11 15 1617 22 29 Placebo N 6 5 5 4 4 4 4 4 3 4 3 Median 6.45 6.25 6.25 6.52 6.426.28 6.58 6.51 6.64 6.35 6.61 Mean 6.45 6.28 6.40 6.48 6.36 6.34 6.546.52 6.50 6.40 6.40 StdErr 0.25 0.12 0.15 0.18 0.24 0.16 0.11 0.19 0.310.23 0.30 50 mg N 10 10 10 10 10 10 10 10 10 10 10 Median 6.81 6.69 6.586.55 6.56 6.46 6.57 6.45 6.54 6.73 6.67 Mean 6.72 6.72 6.60 6.56 6.626.47 6.57 6.57 6.54 6.64 6.71 StdErr 0.11 0.11 0.12 0.06 0.10 0.09 0.080.11 0.08 0.10 0.09 100 mg N 11 10 10 10 9 10 10 9 9 10 4 Median 6.756.93 6.80 6.46 6.59 6.56 6.41 6.40 6.72 6.66 6.71 Mean 6.60 6.68 6.526.43 6.42 6.36 6.30 6.23 6.65 6.53 6.67 StdErr 0.16 0.24 0.23 0.21 0.240.22 0.22 0.23 0.16 0.18 0.17

TABLE 38 Summary Statistics of Change From Baseline (Day 1) in Log₁₀ HCVRNA Day Treatment 2 4 8 11 15 16 17 22 29 Placebo N 5 4 4 4 4 4 3 4 3Median 0.17 0.21 0.15 0.08 0.31 0.21 0.27 0.17 0.09 Mean 0.12 0.22 0.100.08 0.28 0.25 0.15 0.14 0.09 StdErr 0.09 0.12 0.16 0.06 0.15 0.10 0.180.09 0.16  50 mg N 10 10 10 10 10 10 10 10 10 Median −0.07 −0.13 −0.06−0.26 −0.10 −0.13 −0.21 −0.09 −0.04 Mean −0.13 −0.16 −0.11 −0.26 −0.15−0.15 −0.18 −0.09 −0.01 StdErr 0.05 0.07 0.05 0.06 0.08 0.05 0.07 0.060.10 100 mg N 10 10 9 10 10 9 9 10 4 Median −0.12 −0.24 −0.20 −0.28−0.43 −0.49 −0.24 −0.19 −0.12 Mean −0.16 −0.25 −0.21 −0.32 −0.38 −0.39−0.18 −0.15 0.13 StdErr 0.07 0.10 0.16 0.13 0.12 0.14 0.15 0.13 0.28

The clinical evaluation of val-mCyd in the tested patients is shown inFIG. 21. This figure depicts the median change from baseline in Log₁₀HCV RNA by visit.

EXAMPLE 41 Evaluation of Test Compounds

Several of the compounds described herein were tested in the BVDV cellprotection assay described above. FIG. 22 is a table of the EC₅₀ andCC₅₀ of representative compounds in a BVDV cell protection assay, toshow the efficacy of the compounds.

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention, will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of thisinvention. LENGTHY TABLE REFERENCED HERE US20070060541A1-20070315-T00001Please refer to the end of the specification for access instructions.LENGTHY TABLE REFERENCED HERE US20070060541A1-20070315-T00002 Pleaserefer to the end of the specification for access instructions. LENGTHYTABLE REFERENCED HERE US20070060541A1-20070315-T00003 Please refer tothe end of the specification for access instructions. LENGTHY TABLEREFERENCED HERE US20070060541A1-20070315-T00004 Please refer to the endof the specification for access instructions. LENGTHY TABLE REFERENCEDHERE US20070060541A1-20070315-T00005 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00006 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00007 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00008 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00009 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00010 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00011 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00012 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00013 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00014 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00015 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00016 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00017 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00018 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00019 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00020 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00021 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00022 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00023 Please refer to the end of thespecification for access instructions. LENGTHY TABLE REFERENCED HEREUS20070060541A1-20070315-T00024 Please refer to the end of thespecification for access instructions. LENGTHY TABLE The patentapplication contains a lengthy table section. A copy of the table isavailable in electronic form from the USPTO web site(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070060541A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. (canceled)
 2. A method for the treatment of a host infected with aFlaviviridae virus, comprising administering an effective treatmentamount of a compound of formula

or a pharmaceutically acceptable salt thereof wherein: Base is a purinebase, R¹ is hydrogen, mono, di or triphosphate or a stabilizedphosphate; acyl; an amino acid ester; a carbohydrate; a peptide; or apharmaceutically acceptable leaving group which when administered invivo is capable of providing a compound wherein R¹ is independently H orphosphate; and R² is acyl; an amino acid ester; a carbohydrate; apeptide; or a pharmaceutically acceptable leaving group which whenadministered in vivo is capable of providing a compound wherein R² ishydrogen; in combination or alternation with a second antiviral agent.3. The method of claim 2, wherein the virus is hepatitis C. 4-7.(canceled)
 8. The method of claim 2, wherein the compound orpharmaceutically acceptable salt thereof, is in the form of a dosageunit.
 9. The method of claim 8, wherein the dosage unit is a tablet orcapsule.
 10. The method of claim 2, wherein the host is a human.
 11. Themethod of claim 2, wherein the compound is in substantially pure form.12. The method of claim 2, wherein the compound is at least 90% byweight of the β-D-isomer.
 13. The method of claim 2, wherein thecompound or a pharmaceutically acceptable salt thereof is administeredin combination with a pharmaceutically acceptable carrier.
 14. Themethod of claim 13, wherein the pharmaceutically acceptable carrier issuitable for oral delivery.
 15. The method of claim 13, wherein thecompound or a pharmaceutically acceptable salt thereof, is in the formof a dosage unit.
 16. The method of claim 15, wherein the dosage unitcontains 50 to 1000 mg of the compound.
 17. The method of claim 16,wherein said dosage unit is a tablet or capsule.
 18. (canceled)
 19. Themethod of claim 13, wherein the compound or pharmaceutically acceptablesalt thereof, is in substantially pure form.
 20. The method of claim 13,wherein the pharmaceutically acceptable carrier is suitable forsystemic, topical, parenteral, inhalant or intravenous delivery.
 21. Themethod of claim 2, wherein R¹ is hydrogen, mono, di or triphosphate or astabilized phosphate.
 22. The method of claim 2, wherein R² is acyl. 23.The method of claim 2, wherein R² is an amino acid ester.
 24. The methodof claim 22, wherein acyl is of the formula C(O)R′, wherein R′ is astraight, branched, or cyclic alkyl.
 25. The method of claim 2, whereinR¹ is hydrogen.
 26. The method of claim 22, wherein acyl is of theformula C(O)R′ wherein R′ is aryl, alkaryl, aralkyl, alkoxyalkyl oraryloxyalkyl.
 27. The method of claim 22, wherein R² is acetyl.
 28. Themethod of claim 22, wherein R¹ is H.
 29. The method of claim 2, whereinthe base is selected from the group consisting of N⁶-alkylpurine,N⁶-acylpurine, N⁶-benzylpurine, N⁶-vinylpurine, N⁶-acetylenic purine,N⁶-hydroxyalkylpurine, N⁶-alkylaminopurine, N⁶-thioalkylpurine,N²-alkylpurine, N²-alkyl-6-thiopurine and C⁵-hydroxyalkyl purine. 30.The method of claim 2, wherein the base is adenine.
 31. The method ofclaim 2, wherein the base is guanine.
 32. The method of claim 2, whereinR² is an ester of an amino acid selected from the group consisting ofglycine, alanine, valine, leucine, isoleucine, methionine,phenylalanine, tryptophan, proline, serine, threonine, cysteine,tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginineand histidine.
 33. The method of claim 2, wherein R² is an ester of anaturally occurring or synthetic α, β, γ, or δ amino acid.
 34. Themethod of claim 2, wherein R² is an ester of an amino acid in the Lconfiguration.
 35. The method of claim 2, wherein R² is an ester ofvaline.
 36. The method of claim 2, wherein the host is human.
 37. Themethod of claim 23, wherein the host is human.
 38. The method of claim2, wherein: R¹ is H; and R² is acyl or an amino acid ester.
 39. Themethod of claim 38, wherein the base is selected from the groupconsisting of N⁶-alkylpurine, N⁶-acylpurine, N⁶-benzylpurine,N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-hydroxyalkylpurine,N⁶-alkylaminopurine, N⁶-thioalkylpurine, N²-alkylpurine,N²-alkyl-6-thiopurine and C⁵-hydroxyalkyl purine.
 40. The method ofclaim 38, wherein R² is an amino acid ester.
 41. The method of claim 38,wherein the base is adenine.
 42. The method of claim 38, wherein thebase is guanine.
 43. The method of claim 38, wherein R² is an ester ofan amino acid selected from the group consisting of glycine, alanine,valine, leucine, isoleucine, methionine, phenylalanine, tryptophan,proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartate, glutamate, lysine, arginine and histidine.
 44. The method ofclaim 38, wherein R² is an ester of a naturally occurring or synthetica, β, γ, or δ amino acid ester.
 45. The method of claim 38, wherein R²is an ester of an amino acid in the L configuration.
 46. The method ofclaim 38, wherein the host is a human.
 47. The method of claim 43,wherein the host is a human.
 48. A method for the treatment of a hostinfected with a Flaviviridae virus, comprising administering aneffective treatment amount of a compound of formula:

or a pharmaceutically acceptable salt thereof wherein: Base is a purinebase; R¹ is H or phosphate; R² and R³ are independently H, phosphate,acyl or an amino acid ester, wherein at least one of R² and R³ is acylor an amino acid ester; in combination or alternation with a secondanti-viral agent.
 49. The method of claim 2, wherein the secondanti-viral agent is selected from the group consisting of ribavirin, aninterleukin, a helicase inhibitor, a nucleoside, and an inhibitor ofIRES-dependent translation.
 50. The method of claim 2, wherein thesecond anti-viral agent is a polymerase inhibitor.
 51. The method ofclaim 2, wherein the second anti-viral agent is an NS3 proteaseinhibitor.
 52. The method of claim 2, wherein the second anti-viralagent is an interferon.
 53. The method of claim 52, wherein the secondagent is selected from the group consisting of pegylated interferonalpha 2a, interferon alphacon-1, natural interferon, albuferon,interferon beta-1a, omega interferon, interferon alpha and interferongamma-1b.
 54. The method of claim 10, wherein the virus is hepatitis C.55. The method of claim 2, wherein the base is hypoxanthine.
 56. Themethod of claim 2, wherein the base is 2,6-diaminopurine.
 57. The methodof claim 2, wherein the base is 6-chloropurine.
 58. The method of claim49, wherein the virus is hepatitis C.
 59. The method of claim 50,wherein the virus is hepatitis C.
 60. The method of claim 51, whereinthe virus is hepatitis C.
 61. The method of claim 52, wherein the virusis hepatitis C.
 62. The method of claim 53, wherein the virus ishepatitis C.
 63. The method of claim 58, wherein the host is human. 64.The method of claim 59, wherein the host is human.
 65. The method ofclaim 60, wherein the host is human.
 66. The method of claim 61, whereinthe host is human.
 67. The method of claim 62, wherein the host ishuman.